bioprobes 75 journal of cell biology applications · bioprobes 75. journal of cell biology...

BIOPROBES 75JOURNAL OF CELL BIOLOGY APPLICATIONS MAY 2017

ALSO FEATURINGComprehensive strategy for antibody validationSuper Bright fluorescent polymer dyes for the violet laserPrimeFlow RNA assay for detecting RNA targets by flow cytometry

Amplify signals for fluorescence imaging

SuperBoost kits with Alexa Fluor tyramides

Congratulations to the winners of the Cell-ebrate Science Imaging Contest

We asked scientists in Singapore, Taiwan, and the Southeast Asia region to send us cell images showcasing their research using Invitrogen™ fluorescent reagents, and the submissions were stunning. After careful review by a panel of Thermo Fisher Scientific researchers, all experts in the field of fluorescence microscopy, three winning images were chosen.

1st prize winnerJaron Liu, Institute of Medical Biology, SingaporeFilopodia-like protrusions in N1E-115 neuroblastoma cells. The nanoscale architecture of filopodia-like protrusions in N1E-115 neuroblastoma cells was investigated using Invitrogen™ Alexa Fluor™ 568 Phalloidin (pseudocolored blue-green) and state-of-the-art super-resolution microscopy. The image was acquired on a DeltaVision OMX™ 3D-SIM with 100x 1.4NA lens, 3 µm stack, 125 nm per stack, maximum projection, and an image pixel size of 37.5 nm.

3rd prize winnerJonathan Aow, National University of Singapore, SingaporeGFP-expressing primary hippocampal neuron. Primary hippocampal neurons were cultured for 15 days, transfected with GFP (green), and then fixed and stained 2 days later. Axons were labeled with anti–NF-L antibody and Alexa Fluor™ 405 secondary antibody (blue), and the somato-dendritic compartment was labeled with anti-MAP2A/B antibody and Invitrogen™ Alexa Fluor™ 647 secondary antibody (red); neighboring untransfected neurons are visible. Images were acquired on a Zeiss™ LSM700 confocal microscope with a 63x objective.

2nd prize winnerChan Jia Pei, Duke-NUS Medical School, SingaporePsychedelic cerebrum. A coronal section of a mouse brain was labeled with antibody markers for myelin (green, Invitrogen™ Alexa Fluor™ 488 secondary antibody), axons (red, Invitrogen™ Alexa Fluor™ 555 secondary antibody), and microglia (magenta, Invitrogen™ Alexa Fluor™ 633 secondary antibody); nuclei were stained with Hoescht™ 33342 dye (blue). The fluorescent markers highlight the structure of the cortex, hippocampus, striatum, thalamus, and hypothalamus in this section.

For Research Use Only. Not for use in diagnostic procedures. © 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. DeltaVision OMX is a trademark of GE Healthcare Bio-Sciences Corporation. Hoechst is a trademark of Hoechst GmbH. Zeiss is a trademark of Carl Zeiss AG Corporation.

Congratulations to our first, second, and third place winners for their brilliant “beautiful science” images.

Explore our imaging resources at thermofisher.com/cellimaging

BIOPROBES 74This issue focuses on CRISPR-

Cas9–based research, including

the combinat ion of genome

editing and functional proteomics

as well as a description of our

comprehens ive por t fo l io of

reagents for Cas9 delivery through cell function

assays. Also highlighted are several stem cell studies

and fluorescence-based tools for flow cytometry and

imaging, such as the FluxOR™ II Green K+ channel assay.

Cover imageFilopodia-like protrusions in N1E-115 neuroblastoma cells. The nanoscale architecture of filopodia-like protrusions in N1E-115 neuroblastoma cells was investigated using Invitrogen™ Alexa Fluor™ 568 Phalloidin (Cat. No. A12380) and state-of-the-art super-resolution microscopy. The image was acquired on a DeltaVision OMX™ 3D-SIM with 100x 1.4NA lens, 3 µm stack, 125 nm per stack, maximum projection, and an image pixel size of 37.5 nm. Image contributed by Jaron Liu, Institute of Medical Biology, Singapore.

Previous issues

BIOPROBES 72I n th is issue, we descr ibe

hypoxia research tools, 3D

cancer spheroid studies, and

the multiplexable Click-iT™ Plus

TUNEL apoptosis assays, as well

as the CellInsight™ CX7 High-

Content Analysis Platform. We also highlight ProLong™

Live Antifade Reagent for live-cell imaging, and

ProLong™ Diamond and SlowFade™ Diamond Antifade

Mountants for fixed-cell imaging.

BIOPROBES 72MOLECULAR PROBES JOURNAL OF CELL BIOLOGY APPLICATIONS

NOVEMBER 2015

SPECIAL IMAGING ISSUE

Visualize apoptosis in context

with Click-iT Plus TUNEL assays

ALSO FEATURING

Intracellular detection of hypoxiaFormation of uniform 3D cancer spheroidsProtection from photobleaching for live cells

Thermo Fisher Scientific5781 Van Allen Way

Carlsbad, California 92008

United States

Tel: +1 760 603 7200

Toll-Free Tel: 800 955 6288

Fax: +1 760 603 7229

Email: [email protected]

Back issues of BioProbes Journal published after 1995 are available at thermofisher.com/bioprobes. Subscribe to BioProbes Journal at thermofisher.com/subscribebp.

For Research Use Only. Not for use in diagnostic procedures.© 2017 Thermo Fisher Scientific Inc. All rights reserved. The trademarks mentioned herein are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

BD Horizon and Brilliant Violet are trademarks of Becton, Dickinson and Company. Cell-Mate3D is a trademark of BRTI Life Sciences Inc. cOmplete is a trademark of Roche Diagnostics GMBH.

DeltaVision OMX is a trademark of GE Healthcare Bio-Sciences Corp. FLICA is a trademark of Immunchemistry Technologies LLC. GraphPad Prism is a trademark of GraphPad Software, Inc. Hoechst

is a trademark of Hoechst GmbH. Luminex is a trademark of Luminex Corp. Nikon and Eclipse are trademarks of Nikon Instruments Inc. PhiPhiLux is a trademark of Oncolmmunin Inc. SIGMAFAST is a

trademark of Sigma-Aldrich Co. Sprague Dawley is a trademark of Envigo. Taxol is a trademark of Bristol-Myers Squibb Company. Triton is a trademark of The Dow Chemical Company or an affiliated

company of Dow. TSA is a trademark of PerkinElmer Life Sciences, Inc. VioBlue and VioGreen are trademarks of Miltenyi Biotec GmbH. Zeiss is a trademark of Carl Zeiss AG Corp.

COL03855 0517

thermofisher.com/bioprobes

BIOPROBES 73This issue features Click-iT™ EdU

and TUNEL colorimetric assays

for immunohistochemistry, as

well as Superclonal™ recombinant

secondary ant ibodies. A lso

discussed are high-speed cell

counting and generational tracing assays by flow

cytometry, the TurboLuc™ Luciferase One-Step Glow

Assay, and the SureCast™ Handcast and Novex™

WedgeWell™ precast protein gel systems.

Thermo Fisher Scientific

4401 E Marginal Way S

Seattle, WA 98134

Antibodies seeking partners for binding relationshipsOur antibodies and your antigens should get together

Find out more at thermofisher.com/antibodies

For Research Use Only. Not for use in diagnostic procedures. © 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

Over 74,000 functionally tested Invitrogen™ antibodies, covering 85% of the proteome, ready to bond.

Don’t be shy, meet your match—it’s guaranteed.*

* Terms and conditions apply. For complete details, go to thermofisher.com/antibody-performance-guarantee

BioProbes 75

thermofisher.com/bioprobes • May 2017

EditorsMichelle SpenceGrace Richter

DesignerKim McGovernLynn Soderberg

Production ManagerBeth Browne

ContributorsParinaz AliahmadLaura AllredBrian AlmondKris BarnetteDan BeachamAlex BehlingJoy BickleLisa BirkbyJolene BradfordBeth BrowneJohn BucciSuzanne BuckGayle BullerWayne ConsidineJeff CroissantWilliam DietrichMelanie DowdHelen FleisigLeigh FosterCastle FunatakeJane HelmerKamran JamilWilliam KangKevin KeppleSteve LaiHong Van LeDylan MalayterJeffrey MonetteMonica O’Hara-NoonanBhavin PatelGreg PottsJohn RogersPatricia SardinaLaura ShapiroMatt SlaterDariusz StepniakMarcy WickettMichelle YanZhen Yang

Published by Thermo Fisher Scientific Inc. © 2017

BioProbes Journal, available in print and online at thermofisher.com/bioprobes, is dedicated to providing researchers with the very latest information about cell biology products and their applications. For a complete list of our products, along with extensive descriptions and literature references, please see our website.

ONLINE AND ON THE MOVE

2 | Behind the Bench blog, Molecular Probes School of Fluorescence, Antibodies Learning Center, mass spectrometry resources, and more

JUST RELEASED

4 | Our newest cellular analysis products and technologies

TOOLS FOR FLUORESCENCE IMAGING

7 | Amplify signals for fluorescence imagingSuperBoost kits with Alexa Fluor tyramides for signal amplification

11 | Cancer biology in the third dimensionFluorescent reagents for staining 3D cell cultures created with Cell-Mate3D matrix

RESEARCH WITH ANTIBODIES

13 | Harness immune checkpoints to combat tumorsImmune checkpoint antibodies for flow cytometry, IHC, and functional bioassays

16 | Comprehensive strategy for antibody validationCharacterization of antibody performance using immunoprecipitation and mass spectrometry

19 | Examine signaling pathways with targeted proteomicsQuantitative analysis of the AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry

TOOLS FOR FLOW CYTOMETRY

22 | Bright fluorescent polymer dyes for the violet laserSuper Bright antibody conjugates

25 | Powering up drug discoveryCancer biology applications using the Attune NxT Flow Cytometer

28 | Evaluate both RNA and protein targets in single cellsPrimeFlow RNA assay for detecting RNA targets by flow cytometry

JOURNAL CLUB

30 | Coming ’round to spheroid culture

32 | Measurement and characterization of apoptosis by flow cytometry

CENTER INSERT

| Fluorophore and reagent selection guide for flow cytometry

Scott Brush 1

Yi Wen Chai 1

Caitlin Johnson 1

Eu Han Lee 1

Beth Lindborg 1

1 BRTI Life Sciences, Two Harbors, Minnesota

2 | thermofisher.com/bioprobes © 2017 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.

ONLINE AND ON THE MOVE BIOPrOBEs 75

At Thermo Fisher Scientific, we are committed to furthering your scientific advances by providing

a comprehensive suite of products for the analysis of cells and their functions. The addition

of the eBioscience™ portfolio of flow cytometry antibodies and reagents to the innovative

Invitrogen™ flow cytometry reagents and instruments enables us to better provide the tools

your experiments demand. We are fully committed to maintaining product quality while also

making the transition as seamless as possible for you. Beyond our products, we also provide

dedicated service and support teams and educational resources to help support your studies.

From antibodies and functional reagents to instrumentation, Thermo Fisher Scientific is now

poised to more fully serve your research needs. We are focused on advancing meaningful

discoveries and partnering to make tools for cellular analysis effective, affordable, and widely

accessible for all life scientists. To learn more about our wide-ranging array of flow cytometry

products, visit thermofisher.com/flowcytometry.

Do you work with a flow cytometer? Do you want to learn more from experienced scientists

in the field? Thermo Fisher Scientific now has an archive of flow cytometry articles available

on the “Behind the Bench” blog. Learn about “Flow cytometer fluidics and why they are not all

the same” from Greg Kaduchak, PhD, Thermo Fisher Scientific R&D Engineer. Studying cell

proliferation using CFSE or BrdU? We have the blog for you! Get advice from Andrew Filby,

PhD, Flow Cytometry Core Facility Director and ISAC SRL-Emerging Leader at Newcastle

University, UK, or Bill Telford, PhD, Flow Cytometry Research Core Manager at the National

Cancer Institute, NIH, Bethesda, Maryland, to help you get the most from your CFSE- or BrdU-

based cell proliferation assays. Read all about it at thermofisher.com/flowblog.

We are pleased to announce the release of several eLearning modules for protein and cell

analysis applications. Available in the new and improved Protein and Cell Analysis Education

virtual environment, these self-paced animated courses include knowledge checks throughout

and a practical applications component to help you see what you have learned. The first

courses cover:

■ T cell stimulation and proliferation: Part 1 and Part 2

■ Protein sample preparation—Techniques and applications: Part 1 and Part 2

All content is available 24 hours a day, 7 days a week, and is viewable from the convenience of

your desk, tablet, or mobile device. See what it’s all about at thermofisher.com/elearningcourses.

eLearning modules for protein and cell analysis

Behind the Bench blog goes with the flow

Strengthening our flow cytometry portfolio: eBioscience antibodies

thermofisher.com/bioprobes | 3 © 2017 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.

BIOPrOBEs 75 ONLINE AND ON THE MOVE

Invitrogen™ primary and secondary antibodies represent a large and ever-improving portfolio

of products designed to help researchers identify, locate, quantitate, and purify proteins and

other biomolecules. Selecting individual antibody products from among hundreds of options can

be a challenge. The Thermo Fisher Scientific Antibodies Learning Center contains educational

materials designed to empower researchers and technicians with the background information

about immunotechnologies that is necessary to develop, select, and use antibodies to advance

their research. Learn more at thermofisher.com/ablearning.

The module “Fluorescence Basics” is now available online at the Molecular Probes School of

Fluorescence. It covers the fundamentals of fluorescence, from the process of fluorescence

and photobleaching to the anatomy of a fluorescence spectrum and how filters work, and is

brought to you by the scientists who created the Molecular Probes Handbook and the “Imaging

Basics” module. Check it out today at thermofisher.com/mpsf-fluorescence.

Now available upon request, the protein purification tools poster provides helpful information

about the Thermo Scientific™ and Invitrogen™ portfolios of magnetic beads and magnetic

agarose, as well as Superflow and POROS™ resins for applications ranging from small-scale

high-throughput screening to process-scale purification. This poster features practical informa-

tion, including binding capacity, maximum flow rates, and the recommended application scale

and packaging options for each product. Key upstream and downstream products are also

highlighted as part of the integrated protein purification workflows. Request your free poster

today (see terms and conditions) at thermofisher.com/purificationposter.

The Mass Spectrometry Resources Center provides valuable tools to help with your mass

spectrometry experiments, including a free 166-page technical handbook, Protein sample

preparation and quantitation for mass spectrometry. In addition, you’ll have access to helpful

white papers and late-breaking posters for specific applications like subcellular fractionation,

crosslinking, peptide fractionation, isobaric labeling, and more. You can also hear from our

experts on sample preparation and targeted proteomics workflows through our on-demand

webinars. Register now at thermofisher.com/msresources.

The newest Molecular Probes School of Fluorescence module: Fluorescence Basics

Mass spectrometry technical handbook now available

Antibodies Learning Center now online

Protein purification tools poster available upon request


JUsT rELEAsED BIOPrOBEs 75

Invitrogen™ Alexa Fluor™ Plus secondary antibodies are designed

to help you detect low-abundance targets in rare or critical samples

using fluorescence imaging or western blotting. The Alexa Fluor

antibodies you rely on for superior brightness and photostability are

now available in an exclusive formulation using proprietary “plus” dye

chemistry, which provides up to 4 times higher signal-to-noise ratios.

This unique formulation, coupled with stringent pre-adsorption protocols

to remove antibodies that bind nonspecifically, enables Alexa Fluor Plus

antibodies to offer enhanced sensitivity and minimal cross-reactivity

for those challenging samples. See our complete line of Alexa Fluor

Plus secondary antibodies at thermofisher.com/alexafluorplus.

Improved signal-to-noise ratios with Alexa Fluor Plus antibodies reveal greater

detail. Fixed and permeabilized E18 Sprague Dawley™ primary cortical neuronal cells

were labeled with anti–β-III tubulin primary antibody and (A) Invitrogen™ Alexa Fluor™

488 or (B) Invitrogen™ Alexa Fluor™ Plus 488 (Cat. No. A32723) secondary antibod-

ies. Cells were then counterstained with Hoechst™ 33342 dye (Cat. No. H3570) and

imaged on a Zeiss™ LSM 710 inverted confocal microscope at 40x magnification.

Enhanced Alexa Fluor secondary antibodies: Higher signal-to-noise ratios for hard-to-detect targets

Thermo Scientific™ Pierce™ protease and phosphatase inhibitor

tablets are ideal for the protection of proteins during extraction pro-

cedures or lysate preparation using primary cells, cultured mammalian

cells, animal tissues, plant tissues, yeast cells, or bacterial cells.

With improved formulations, these ready-to-use inhibitor tablets

dissolve quickly into a clear solution and are directly compatible with

Thermo Scientific™ Pierce™ BCA Protein Assays. Find out more at

thermofisher.com/inhibitorcocktails.

Improved formulation for Pierce protease and phosphatase inhibitor tablets

Performance comparison between three commercially available protease inhib-

itor tablets. Pancreatic extract (100 μL, 0.5 μg/μL) was incubated with cleavable

fluorogenic substrates for trypsin and cysteine proteases, metalloproteases, and

cathepsins, in the presence of Thermo Scientific™ Pierce™ Protease Inhibitor Mini

Tablets (Cat. No. A32953), Roche cOmplete™ Protease Inhibitor Tablets, and Sigma-

Aldrich SIGMAFAST™ Protease Inhibitor Cocktail Tablets, with and without EDTA.

Reactions were incubated for 1 hr at 37ºC, and fluorescence was determined at the

appropriate detection emissions on a Thermo Scientific™ Varioskan™ Flash microplate

reader. Percent protease inhibition is shown for each protease inhibitor formulation.

Product Quantity Cat. No.

Pierce™ Protease Inhibitor Mini Tablets 30 tablets A32953

Pierce™ Protease Inhibitor Tablets 20 tablets A32963

Pierce™ Protease Inhibitor Mini Tablets, EDTA-free 30 tablets A32955

Pierce™ Protease Inhibitor Tablets, EDTA-free 20 tablets A32965

Pierce™ Phosphatase Inhibitor Mini Tablets 20 tablets A32957

Pierce™ Protease and Phosphatase Inhibitor Mini Tablets 20 tablets A32959

Pierce™ Protease and Phosphatase Inhibitor Mini Tablets, EDTA-free

20 tablets A32961

0

20

60

80

100

120RocheThermo Scienti�c Sigma

40% In

hib

ition

Trypsin protease Metalloproteasesand cathepsins

Cysteine protease

With EDTA With EDTA With EDTAEDTA-free EDTA-free EDTA-free

Protease inhibitors:

Invitrogen™ Novex™ Tris-Glycine Plus Midi Gels are the next generation of precast midi

polyacrylamide gels, providing high-quality performance and separation of proteins into well-

resolved bands using Laemmli electrophoresis technology. Available in fixed (10%, 12%) and

gradient (4–12%, 4–20%, 8–16%) concentrations and with different well formats (12+2 wells,

20 wells, 26 wells), these Tris-glycine gels feature a longer shelf life (up to 1 year at 4°C), fast

run conditions (<60 minutes at constant voltage), and the flexibility to run native or denatured

protein samples. See our complete selection of precast midi gels at thermofisher.com/midigels. Novex Tris-Glycine Plus Midi Gel System.

New and improved Tris-glycine midi protein gels


BIOPrOBEs 75 JUsT rELEAsED

For over 50 years, the alamarBlue dye has proven to be a reliable

reagent for measuring cell viability in bacteria, yeast, fungi, protozoa,

and cultured mammalian cells. This resazurin-based redox indicator

is converted into a fluorescent and colorimetric product (resorufin) by

metabolically active cells. Damaged and nonviable cells have lower

innate metabolic activity and generate proportionally lower signals.

The Invitrogen™ alamarBlue™ Cell Viability Reagent is a mixture

of salts, a background suppressor, and resazurin—a formulation that

accurately and reliably reports changes in redox potential. Because of

the highly sensitive nature of the alamarBlue reagent, the manufacturing

processes need to be well controlled. In the absence of proper manu-

facturing processes, the background fluorescence signal of the reagent

could increase, causing a decrease in the signal-to-background ratio

and a reduction in assay performance. Our recently improved manu-

facturing process has resulted in a product with superior performance,

displaying >50% reduction in background fluorescence compared with

the previously manufactured material or another commercially available

product. Discover the quality of the improved alamarBlue Cell Viability

Reagent at thermofisher.com/alamarblue.

Improvements to the alamarBlue cell viability and cytotoxicity reagent


alamarBlue™ Cell Viability Reagent 25 mL DAL1025

alamarBlue™ Cell Viability Reagent 100 mL DAL1100

Bac

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und

�uo

resc

ence

5

0

10

15

20

Bio-Radproduct

Newmanufacturing

process

Previousmanufacturing

process

5

0

10

15

20

Bio-Radproduct

Newmanufacturing

process

Previousmanufacturing

process

Sig

nal-

to-b

ackg

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d r

atio

BA

Evaluation of our new manufacturing process for the alamarBlue reagent.

(A) Comparison of the background fluorescence from various alamarBlue products

reveals that the improved Invitrogen™ alamarBlue™ Cell Viability Reagent (Cat.

No. DAL1025) shows the lowest level of background signal. (B) As expected,

the decrease in background fluorescence results in an increase in the signal-to-

noise ratio. The newly manufactured alamarBlue reagent has a significantly higher

signal-to-background ratio than the previously manufactured reagent or another

commercially available product.

The Invitrogen™ FluxOR™ Red Potassium Ion Channel Assay utilizes a

red-fluorescent indicator dye to detect potassium ion channel activity.

Similar to the Invitrogen™ FluxOR™ II Green assay, the FluxOR Red

assay takes advantage of the well-established permeability of potassium

channels to thallium ions. When potassium channels are opened by a

stimulus, extracellular thallium flows down its concentration gradient into

cells and binds to the FluxOR thallium-sensitive dye. In the FluxOR Red

assay, an increase in cytosolic red fluorescence (Ex/Em = 560/590 nm)

serves as a surrogate measurement of the activity of any ion channel

or transport process that allows thallium into cells. The FluxOR Red

Potassium Ion Channel Assay can be configured for either imaging

or high-throughput screening applications and is compatible with

detection of GFP and other green-fluorescent probes including the

FluxOR II Green dye, which can be used as a second, independent

measure of potassium channel activity. Learn more about the FluxOR

potassium ion channel assays at thermofisher.com/fluxor.

Introducing the FluxOR Red Potassium Ion Channel Assay

Multiplexing the FluxOR Red Potassium Ion Channel Assay with a cell viability

assay. Gibco™ Primary Rat Cortex Neurons (Cat. No. A1084001) were incubated with

Invitrogen™ Calcein AM (Cat. No. C3099) and Invitrogen™ FluxOR™ Red dye (Cat.

No. F20018). After stimulation of the potassium channels, cells were imaged on the

Invitrogen™ EVOS™ FL Auto 2 Imaging System (Cat. No. AMAFD2000) using (A) a

FITC/Alexa Fluor 488 filter set to detect the green fluorescence of live cells stained with

calcein and (B) a TRITC/Alexa Fluor 555 filter set to detect the red fluorescence of the

FluxOR Red reagent, indicative of potassium ion flux. (C) The overlay of the calcein

and FluxOR staining images confirms that live cells show potassium channel activity.


FluxOR™ II Green Potassium Ion Channel Assay 2 plates10 plates100 plates

F20015F20016F20017

FluxOR™ Red Potassium Ion Channel Assay 2 plates10 plates

F20018F20019


JUsT rELEAsED BIOPrOBEs 75

Invitrogen™ Ready Flow™ reagents are ready-to-use solutions designed to stain cells for

analysis by flow cytometry without calculations, dilutions, or pipetting. These reagents simplify

the most common flow cytometry assays that employ nucleic acid dyes, including dead-cell

identification and cell cycle analysis, and are provided in convenient dropper bottles that can

be stored at room temperature. Simply add 2 drops of Ready Flow reagent per 1 x 106 cells,

incubate, and analyze.

Currently available Ready Flow reagents include a variety of fluorescent nucleic acid

stains that are commonly used as cell viability and cell cycle reagents. With these Ready Flow

reagents, preparing your samples for viability or cell cycle analysis by flow cytometry is quick

and easy. Find the newest Ready Flow formulations for your favorite flow cytometry assays;

visit thermofisher.com/readyflow for up-to-date listings.

FxCycle Violet Ready Flow Reagent

0.1

S

Sub-G1

G2/M

G0/G1

500 1,000

Ti-kcilC

roulF axelA

Ud

E 6

47

101

102

103

104

105

106

Product Lasers for excitation Quantity Cat. No.

FxCycle™ Violet Ready Flow™ Reagent 355 or 405 nm laser 120 assays R37166

Hoechst™ 33342 Ready Flow™ Reagent 355 or 405 nm laser 120 assays R37165

Propidium Iodide Ready Flow™ Reagent 488, 532, or 561 nm laser 120 assays R37169

SYTOX AADvanced™ Ready Flow™ Reagent 488, 532, or 561 nm laser 120 assays R37173

SYTOX™ Green Ready Flow™ Reagent 488 nm laser 120 assays R37168

TO-PRO™ 3 Ready Flow™ Reagent 633 nm laser 120 assays R37170

Ready, set, flow! Ready Flow ready-to-use flow cytometry reagents

Cell cycle analysis with FxCycle Violet Ready

Flow Reagent and the Click-iT EdU Alexa Fluor

647 Flow Cytometry Assay Kit. Jurkat cells (human

T cell leukemia) were pulsed with 10 µM EdU for

2 hr prior to detection of proliferating cells with Alexa

Fluor 647 azide using the Invitrogen™ Click-iT™ EdU

Alexa Fluor™ 647 Flow Cytometry Assay Kit (Cat. No.

C10424). Cells were then stained with 2 drops of

Invitrogen™ FxCycle™ Violet Ready Flow™ Reagent

(Cat. No. R37166) and incubated for 30 min at 25°C.

Data were acquired on an Invitrogen™ Attune™ NxT

Flow Cytometer using a 405 nm laser and 440/50 nm

emission filter. Analysis of the population indicates the

following distribution: apoptotic sub-G1 cells, 3.4%;

G0/G1, 49.1%; S, 33.0%; and G2/M, 14.0%.

A key lipid–protein complex in blood, the human low-density lipoprotein (LDL) is a crucial

component of metabolism that drives the transport of fats throughout the body. It is now possible

to follow—in real time—the receptor-mediated endocytosis and trafficking of the LDL complex

in live cells using Invitrogen™ pHrodo™ Red and pHrodo™ Green LDL conjugates. Though dimly

fluorescent at neutral pH outside of cells, pHrodo LDL conjugates fluoresce brightly during and

after internalization as endosomes are increasingly acidified through the endocytic pathway.

With a simple workflow (add the pHrodo LDL conjugate to serum-starved cells), you can

clearly distinguish cell surface–bound vs. internalized pHrodo LDL complexes by measuring

cell fluorescence using fluorescence microscopy, flow cytometry, or high-content analysis. The

pHrodo LDL conjugates are multiplexable with other spectrally compatible fluorescent reagents.

Visit thermofisher.com/phrodo for more information about these effective pH indicators.

Follow LDL uptake and trafficking using pHrodo Red and pHrodo Green LDL conjugates


Image-iT™ Low-Density Lipoprotein Uptake Kit, pHrodo™ Red 1 kit I34360

Low Density Lipoprotein From Human Plasma, pHrodo™ Green conjugate (pHrodo™ Green LDL, 1 mg/mL)

200 µL L34355

Low Density Lipoprotein From Human Plasma, pHrodo™ Red conjugate (pHrodo™ Red LDL, 1 mg/mL)

200 µL L34356

Unlabeled Low Density Lipoprotein From Human Plasma (LDL, 2.5 mg/mL) 200 µL L3486

Unlabeled LDLNo pretreatment Heparin

Endocytosis of pHrodo Red LDL. HepG2 cells

were plated onto poly-D-lysine–coated glass-bottom

dishes, incubated in growth medium for 24 hr at

37°C, then switched to Gibco™ FluoroBrite™ DMEM

(Cat. No. A1896702) with 0.3% BSA. After overnight

incubation, cells were pretreated with vehicular control

(0.3% BSA, left), 250 µg/mL LDL (Cat. No. L3486,

center), or 250 µg/mL heparin (right) for 30 min.

Invitrogen™ pHrodo™ Red LDL (Cat. No. L34356)

was added to the medium at 10 µg/mL, and cells

were further incubated for 3 hr, counterstained with

Invitrogen™ NucBlue™ Live ReadyProbes™ Reagent

(Cat No. R37605), washed twice, and imaged using

the Invitrogen™ EVOS™ FL Auto 2 Imaging System (Cat.

No. AMAFD2000). Images were matched for gain and

exposure. In cells without pretreatment, the endocyto-

sed pHrodo Red LDL complex (red) appears adjacent

to the blue-fluorescent nuclei; in cells pretreated with

unlabeled LDL or heparin, the pHrodo Red LDL remains

outside of the cell and only dimly fluorescent.


BIOPrOBEs 75 TOOLs FOr FLUOrEsCENCE IMAGING

Amplify signals for fluorescence imagingSuperBoost kits with Alexa Fluor tyramides for signal amplification.

Many functionally important proteins such as transcription factors and cell-surface cytokine receptors have

native expression levels far below the detection threshold of labeled primary and secondary antibodies. Not

only do cell signaling proteins vary in abundance by at least 7 orders of magnitude (~101 to 108 copies per

cell), but their distribution within the cell is neither spatially nor temporally static. The Invitrogen™ SuperBoost™

kits with Alexa Fluor™ tyramides provide a highly sensitive method for detecting low-abundance targets

in immunocytochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization (ISH) applications.

Tyramide SuperBoost technology combines the brightness of Alexa Fluor dyes with poly-HRP–mediated

tyramide signal amplification to discern signal from noise (Figure 1), yielding precision and sensitivity

10–200 times greater than that of standard ICC/IHC/ISH methods and 2–10 times greater than that of

other tyramide amplification techniques, including traditional TSA™ methods.

Figure 1 (above). Detection of phosphorylated epidermal growth factor receptor (EGFR) using the Alexa Fluor 488 Tyramide SuperBoost Kit. A549 cells were

incubated with epidermal growth factor (EGF) for 2 min to induce phosphorylation of EGFR on cell-membrane surfaces. Cells were fixed, permeabilized, and blocked

according to the kit protocol. Phosphorylated EGFR was then labeled with rabbit anti-pEGFR primary antibody (clone Y1068) and detected using the Invitrogen™ Alexa

Fluor™ 488 Tyramide SuperBoost Kit with goat anti–rabbit IgG antibody (green, Cat. No. B40922). Total EGFR (phosphorylated and unphosphorylated) was labeled with

anti-EGFR primary antibody (Cat. No. MA5-13070) and detected with Invitrogen™ Alexa Fluor™ 594 secondary antibody (red, Cat. No. R37121). Nuclei were labeled with

Invitrogen™ NucBlue™ Fixed Cell ReadyProbes™ Reagent (blue, Cat. No. R37606). Cells were mounted with Invitrogen™ ProLong™ Diamond mountant (Cat. No. P36961)

and imaged on an inverted confocal microscope.


TOOLs FOr FLUOrEsCENCE IMAGING BIOPrOBEs 75

Tyramide signal ampl i f icat ion is an

enzyme-mediated detection method that

utilizes the catalytic activity of horseradish

peroxidase (HRP) to generate high-density

labeling of a target protein or nucleic acid

sequence in situ [1]. In the first step of this

process, a probe binds to the target via immu-

noaffinity (proteins) or hybridization (nucleic

acids) and is then detected with an HRP-

labeled secondary antibody or streptavidin

conjugate. Next, multiple copies of a labeled

tyramide (e.g., a fluorescent Alexa Fluor tyr-

amide in the SuperBoost kits) are activated

by enzymatic reaction with HRP. Lastly, the

highly reactive, short-lived tyramide radicals

covalently couple with residues (principally the

phenol moiety of protein tyrosine residues) in

the vicinity of the HRP–target interaction site,

resulting in minimal diffusion-related loss of

signal localization (Figure 2).

SuperBoost amplification provides greater sensitivity for hard-to-detect targetsThe tyramide signal amplification technique

used in the SuperBoost kits utilizes the cata-

lytic activity of horseradish peroxidase (HRP)

for high-density labeling of a target protein or

nucleic acid sequence in situ. The enhanced

sensitivity of the SuperBoost kits over other

tyramide signal amplification technologies is

in part due to improvements in the reagents

for each amplification step. Figure 1 shows

an example of the sensitivity of Tyramide

SuperBoost technology to detect phosphory-

lated epidermal growth factor receptor (EGFR)

in the cell membranes of A594 cells, just

2 minutes after treating cells with epidermal

growth factor (EGF) and prior to receptor

internalization.

The Tyramide SuperBoost kits boost

the specific signal by utilizing Alexa Fluor

tyramides, which react with HRP to deposit brightly fluorescent and photostable Alexa Fluor dyes

on surrounding protein tyrosine residues and other similar molecules. In a study of cell-surface

microdomains that serve as attachment sites for Kaposi’s sarcoma–associated herpesvirus

(KSHV), Garrigues and coworkers reported that tyramide signal amplification with several differ-

ent Alexa Fluor tyramides was 5 times more sensitive than conventional immunofluorescence

in their experiments [2].

Furthermore, unlike traditional TSA kits, Tyramide SuperBoost kits employ poly-HRP–

conjugated secondary antibodies (streptavidin is conjugated with standard HRP). Poly-HRP

antibodies contain several HRP enzymes conjugated with short polymers, enhancing the signal

several-fold over standard HRP antibodies (Figure 3); the molar ratio of enzyme to antibody is

approximately 4. Importantly, the poly-HRP antibody is structured in such a way that the conjugate

can penetrate cells or tissue as efficiently as do standard HRP antibodies.

Tyramide SuperBoost kits also employ several strategies to reduce background fluorescence.

First, these kits contain highly cross-adsorbed secondary antibodies to help ensure specificity for

the target primary antibody with minimal cross-labeling of other antibody species. For example,

the poly-HRP–conjugated goat anti–mouse IgG exhibits no detectable reactivity to mouse serum

proteins or IgG from bovine, goat, human, rabbit, or rat. Likewise, the poly-HRP–conjugated goat

anti–rabbit IgG does not bind to rabbit serum proteins or IgG from bovine, goat, human, mouse,

or rat. Second, an optimized blocking buffer is included to help prevent nonspecific binding and

to significantly reduce endogenous peroxidase activity. And third, the Tyramide SuperBoost kits

contain a reagent for preparing an HRP stop solution. Like any enzyme system, it is possible to

overdevelop the signal, which can increase background levels. The HRP stop solution can be

used to obtain maximum signal without an increase in background levels, which is especially

important when amplifying signals localized to fine structures. Images produced with optimized

HRP reaction times are as sharp as images produced with standard ICC/IHC/ISH methods but

are 10 to 200 times more sensitive.

Figure 2. Tyramide signal amplification applied to the immunolabeling of an antigen. The antigen is detected

by a primary antibody (blue) (A), followed by a poly–horseradish peroxidase (poly-HRP) conjugated secondary

antibody (yellow) (B). Activation of the dye-labeled tyramide (green) by HRP results in localized deposition of the

activated tyramide derivative (pink) (C).

HH2O2

2O2

A

HRP

Reactive�uorophore

Unreactive�uorophore

HRP-conjugatedantibody

Primaryantibody

HRP

HRP

B

C



SuperBoost amplification requires less primary antibodyCompared to conventional immunofluorescence techniques, the

Tyramide SuperBoost kits require much smaller amounts of primary

antibody—a tenth to a hundredth as much—to achieve sensitive

detection of target molecules. Even with much less primary antibody,

the Tyramide SuperBoost kits provide detection sensitivities similar

to or greater than those obtained with a fluorescently labeled sec-

ondary antibody in an ICC application (Figure 3). Using less antibody

per experiment will save on the cost of primary antibodies, a major

expense in ICC and IHC workflows. Also, more experiments can be

accomplished using a single vial of primary antibody. Given that some

primary antibodies show significant lot-to-lot variation, using a single

lot throughout a project can produce more reliable results that are

consistent from experiment to experiment.

SuperBoost amplification is multiplexableThe Tyramide SuperBoost kits are available with four spectrally distinct

Alexa Fluor tyramides and three different HRP secondary reagents

(Table 1). This range of choices allows high-resolution detection and

visualization of multiple signals in a single cell or tissue sample. In

addition to multiplex detection using primary antibodies from different

species (Figures 4A and 4B), Tyramide SuperBoost amplification is

also compatible with experiments that use GFP and RFP fusions as

reporters of gene expression (Figure 4C).

Figure 3. Sensitivity of Tyramide SuperBoost kits and other immunodetection

methods using different amounts of primary antibody. HeLa cells were fixed

and permeabilized with the Invitrogen™ Image-iT™ Fixation/Permeabilization Kit (Cat.

No. R37602). Prohibitin was labeled with various dilutions of rabbit anti-prohibitin

primary antibody; the manufacturer recommendation was 1:150 dilution or 5 μg/mL.

Anti-prohibitin antibody was then detected using the Invitrogen™ Alexa Fluor™ 488

Tyramide SuperBoost™ Kit with goat anti–rabbit IgG antibody (Cat. No. B40922), the

Invitrogen™ TSA™ Kit #12 with HRP goat anti–rabbit IgG antibody and Alexa Fluor™

488 tyramide (Cat. No. T20922), or Invitrogen™ Alexa Fluor™ 488 goat anti–rabbit

IgG secondary antibody (Cat. No. A11008). Cells were imaged on the Invitrogen™

EVOS™ FL Auto Imaging System (Cat. No. AMAFD1000) using the same exposure

and gain. These images (bottom) and the quantitation of fluorescence (top) indicate

that the Alexa Fluor 488 Tyramide SuperBoost Kit is more sensitive than both the

TSA kit and directly labeled secondary antibody. At this exposure and gain setting,

prohibitin is not detectable with standard ICC methods.

Figure 4. Multiplexing Tyramide SuperBoost kits with other fluorescent probes. Prior to immunodetection, HeLa cells were fixed and permeabilized using the Invitrogen™

Image-iT™ Fixation/Permeabilization Kit (Cat. No. R37602). After staining, nuclei were labeled with Invitrogen™ NucBlue™ Fixed Cell ReadyProbes™ Reagent (blue, Cat. No.

R37606) and then cells were imaged using a Zeiss™ LSM 710 inverted confocal microscope at 63x magnification. (A) Multiplexing Tyramide SuperBoost kits with secondary

antibodies. In fixed and permeabilized HeLa cells, tubulin was labeled with rabbit anti-tubulin primary antibody and detected with Invitrogen™ Alexa Fluor™ 488 goat anti–rabbit

IgG secondary antibody (green, Cat. No. A11008), and ATP synthase was labeled with mouse anti–ATP synthase subunit IF1 (ATPIF1) antibody (Cat. No. A21355) and

detected with Invitrogen™ Alexa Fluor™ 594 Tyramide SuperBoost™ Kit with goat anti–mouse IgG antibody (red, Cat. No. B40915). (B) Multiplexing two Tyramide SuperBoost

kits. In fixed and permeabilized HeLa cells, prohibitin was labeled with rabbit anti-prohibitin primary antibody and detected using the Invitrogen™ Alexa Fluor™ 647 Tyramide

SuperBoost™ Kit with goat anti–rabbit IgG antibody (purple, Cat. No. B40926), and β-catenin was labeled with mouse anti–β-catenin primary antibody (Cat. No. 13-8400)

and detected with Alexa Fluor 488 Tyramide SuperBoost Kit with goat anti–mouse IgG antibody (green, Cat. No. B40912). (C) Multiplexing Tyramide SuperBoost kits with

a GFP reporter protein. Prior to fixation, HeLa cells were treated overnight with Invitrogen™ CellLight™ Peroxisome-GFP (BacMam 2.0, Cat. No. C10604) to produce a

peroxisome-targeted GFP fusion protein (green). Cells were then fixed and permeabilized using the Image-iT Fixation/Permeabilization Kit. Prohibitin was labeled with rabbit

anti-prohibitin primary antibody and detected using the Invitrogen™ Alexa Fluor™ 594 Tyramide SuperBoost™ Kit with goat anti–rabbit IgG antibody (red, Cat. No. B40925).

Primary antibody dilution

1:150 1:600 1:2,4001:300 1:1,200 1:4,8000

600

1,200

1,800Tyramide SuperBoost with Alexa Fluor 488 tyramide

Rel

ativ

e �u

ores

cenc

e

Alexa Fluor 488 secondary antibodyTSA Kit with Alexa Fluor 488 tyramide

1:150 1:600 1:2,4001:300 1:1,200 1:4,800

Tyramide SuperBoost withAlexa Fluor 488 tyramide

TSA Kit withAlexa Fluor 488 tyramide

Alexa Fluor 488secondary antibody



References1. Bobrow MN, Harris TD, Shaughnessy KJ et al. (1989) J Immunol Methods

125:279–285.

2. Garrigues HJ, Rubinchikova YE, Rose TM (2014) Virology 452–453:75–85.

3. Tóth ZE, Mezey E (2007) J Histochem Cytochem 55:545–554.

Table 1. Tyramide SuperBoost kits.

Labeled tyramide Ex/Em *

Cat. No. of Tyramide SuperBoost™ kits †

Goat anti–mouse IgG

Goat anti–rabbit IgG Streptavidin

Alexa Fluor™ 488 495/519 B40912

B40941 (50 slides)

B40922

B40943 (50 slides)

B40932

Alexa Fluor™ 555 555/565 B40913 B40923 B40933

Alexa Fluor™ 594 591/617 B40915

B40942 (50 slides)

B40925

B40944 (50 slides)

B40935

Alexa Fluor™ 647 650/668 B40916 B40926 B40936

Biotin-XX NA B40911 B40921 B40931

* Approximate fluorescence excitation and emission maxima, in nm. † Sufficient reagents are provided for 150 18 mm x 18 mm coverslips using 100 µL per slide in critical incubation steps, except where otherwise noted. Poly-HRP–conjugated antibody and HRP streptavidin, as well as Alexa Fluor tyramides, are also available as stand-alone reagents.

Unlike many other amplification methods, Tyramide SuperBoost

technology allows you to detect multiple targets in tissues using primary

antibodies from the same host species. Tyramide signal amplification

produces highly reactive tyramide radicals that covalently react with

tyrosine residues in the vicinity of the HRP conjugate, which results in

minimal diffusion-related loss of signal and additionally makes it possible

to strip off the primary and secondary antibody without significantly

decreasing the fluorescence intensity of the tyramide deposit. Using

the microwave/citrate buffer method described by Tóth and Mezey [3]

to strip off antibodies, we validated this technique with tissue sections

sequentially labeled with the Tyramide SuperBoost kits and three

different rabbit primary antibodies (Figure 5) or two different rabbit

primary antibodies (Figure 6).

Find out more about Tyramide SuperBoost amplificationTyramide SuperBoost kits are designed for superior signal ampli-

fication, with the definition and clarity needed for high-resolution

fluores cence imaging of low-abundance targets. These kits are

simple to use and easily adapted to standard ICC, IHC, or ISH

experimental protocols using a wide variety of cell and tissue types.

We have tested the performance of the Tyramide SuperBoost kits

using formaldehyde-fixed cells in 2D and 3D culture, formalin-fixed,

paraffin-embedded (FFPE) tissues, and cryosection tissues. Learn more

about tyramide signal amplification and the SuperBoost technology at

thermofisher.com/superboostbp75. ■

Figure 5. Sequential labeling and detection of three different rabbit primary anti-

bodies using the Tyramide SuperBoost kits. A formalin-fixed, paraffin-embedded

(FFPE) rat intestinal section was labeled sequentially with rabbit primary antibodies

against H2B, actin, and Ki-67. Primary antibody detection was performed using three

different Invitrogen™ Alexa Fluor™ Tyramide SuperBoost™ kits. Briefly, tissue samples

underwent heat-induced antigen retrieval in citrate buffer, pH 6 (10 min on high

setting in a pressure cooker) and were then sequentially labeled with rabbit anti-H2B

antibody (detected with the Alexa Fluor 647 Tyramide SuperBoost Kit (green, Cat.

No. B40926)), rabbit anti–smooth muscle actin antibody (detected with the Alexa

Fluor 488 Tyramide SuperBoost Kit (red, Cat. No B40922)), and rabbit anti-Ki67

antibody (detected with the Alexa Fluor 594 Tyramide SuperBoost Kit (blue, Cat. No.

B40925)). In between each antibody labeling, tissue samples were microwaved in

citrate buffer, pH 6, on high power until boiling (~2 min), then microwaved for 15 min

at 20% power, and finally allowed to cool to room temperature before subsequent

labeling with the next rabbit antibody [3]. Image was acquired on a Zeiss™ LSM 710

inverted confocal microscope at 20x magnification.

Figure 6. Same-species immunolabeling with Tyramide SuperBoost kits and

detection of proliferation using the Click-iT EdU assay. Proliferating cells were

detected in an FFPE rat mammary tissue section (derived from an animal that was

pulsed with EdU) using the Invitrogen™ Click-iT™ EdU Alexa Fluor™ 647 Imaging Kit

(pink, Cat. No. C10340). The sample was also labeled with two different rabbit primary

antibodies (anti–Ki-67 and anti–smooth muscle actin) and two Tyramide SuperBoost

kits (Alexa Fluor™ 594 Tyramide SuperBoost™ Kit for Ki-67, shown in white, and Alexa

Fluor™ 488 Tyramide SuperBoost™ Kit for smooth muscle actin, shown in green)

using the microwave/citrate buffer method [3] described in Figure 5. Images were

acquired on a Zeiss™ LSM 710 inverted confocal microscope at 20x magnification.



Cancer biology in the third dimensionFluorescent reagents for staining 3D cell cultures created with Cell-Mate3D matrix.Beth Lindborg, Caitlin Johnson, Yi Wen Chai, Eu Han Lee, and Scott Brush (BRTI Life Sciences); Brian Almond (Thermo Fisher Scientific).

Unlike their 2D tissue culture counterparts, tumors and their associated

microenvironments contain a highly complex and dynamic set of inter-

actions between different cell types [1], multiple chemical gradients [2],

and a variety of extracellular matrix components [3,4]. In addition to the

biochemical reactions that take place, a tumor’s physical character-

istics, including the rigidity or stiffness of the extracellular matrix, can

regulate its physiology [5–7]. The next discoveries in cancer biology

and treatment will require tools that help to create more relevant and

predictive models as well as methods to effectively analyze them.

The combination of 3D cell culture techniques with methods

for analyzing cell health promises to deliver new insights into cancer

mechanisms and new strategies for interrupting or subverting critical

pathways. Here we describe the application of fluorescence-based

assays and protocols to a complex 3D environment, often with only minor

adjustments to exposure time or reagent concentration. Fluorescent

reagents from Thermo Fisher Scientific can be used to assess cell

function—including viability, proliferation, and apoptosis—in response

to various 3D culture conditions and treatments.

Create 3D cultures with Cell-Mate3D matrixCell-Mate3D™ matrix from BRTI Life Sciences is a tissue-like matrix

that offers researchers a biologically relevant and chemically defined

microenvironment for in vitro and in vivo biomedical research. The

Cell-Mate3D matrix is composed of two naturally occurring biopoly-

mers: chitosan, a positively charged polysaccharide derived from

chitin, found in the exoskeleton of crustaceans; and hyaluronic acid,

a linear polysaccharide found in the extracellular matrix of connective,

epithelial, and neural tissues. Hyaluronic acid is typically involved in cell

proliferation, migration, embryonic development, and wound healing.

Figure 1. Detection of viable cells in Cell-Mate3D matrix using the LIVE/DEAD

viability assay. AU565 breast cancer cells growing in Cell-Mate3D matrix were

stained using the Invitrogen™ LIVE/DEAD™ Viability/Cytotoxicity Kit (Cat. No. L3224)

and counterstained using Invitrogen™ NucBlue™ Live ReadyProbes™ Reagent (Cat.

No. R37605). (A) In the untreated sample, live cells fluoresce green and nuclei

fluoresce blue; very few nonviable (red-fluorescent) cells are detected. (B) After

treatment with 0.5% Triton™ X-100 detergent, the sample contains primarily non-

viable (red-fluorescent) cells; the purple color of the cells in the image represent

the overlap of the red- and blue-fluorescent stains. Samples were imaged using an

inverted confocal microscope at 20x magnification.

When Cell-Mate3D Dry Blend is combined with cells resuspended

in Cell-Mate3D Hydration Fluid, the matrix forms instantly through elec-

trostatic interactions between carboxyl groups on the hyaluronic acid

and the amine groups on the chitosan. The resulting poly-electrolytic

complex is a fibrous matrix that exhibits tissue-like stiffness, emulating

the natural cell environment. The Cell-Mate3D matrix enables modeling

of cell migration and proliferation; laboratory techniques such as cell

staining and imaging, flow cytometry, and scanning electron microscopy

can be used with this technology [8].

Determine cell viability in 3D culturesThe Invitrogen™ LIVE/DEAD™ Viability/Cytotoxicity Kit provides a

well-established fluorescence assay for determining viability in a wide

range of animal cells. The LIVE/DEAD viability assay comprises two

fluorescent probes—calcein AM, a cell-permeant esterase substrate,

and ethidium homodimer-1, a cell-impermeant high-affinity nucleic

acid stain—that are used simultaneously to stain cells. We used the

LIVE/DEAD viability assay with 3D cell cultures that were created by

embedding AU565 breast cancer cells into Cell-Mate3D matrix. After the

matrix with embedded cells was incubated for 4 days, one sample was

left untreated and an equivalent sample was treated with 0.5% Triton™

X-100 detergent, which disrupts cell membranes and kills cells. Both

samples were then assayed using the LIVE/DEAD Viability/Cytotoxicity Kit

and the supplied protocol, except that the incubation time was reduced

from 30 to 20 minutes to optimize staining levels in this system. After

counter staining with a blue-fluorescent nucleic acid dye, the samples

were imaged on an inverted confocal microscope (Figure 1). The untreated

sample shows live cells labeled with green fluorescence, whereas the

treated sample shows dead cells labeled with red fluorescence.




LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells

1 kit L3224

CellEvent™ Caspase-3/7 Green Detection Reagent

25 µL100 µL

C10723C10423

Click-iT™ EdU Alexa Fluor™ 488 Imaging Kit

1 kit C10337

NucBlue™ Live ReadyProbes™ Reagent

1 kit R37605

NucBlue™ Fixed Cell ReadyProbes™ Reagent

1 kit R37606

References1. Baker BM, Chen CS (2012) J Cell Sci 125:3015–3024.

2. Hoffman BD, Grashoff C, Schwartz MA (2011) Nature 475:316–323.

3. Kimlin LC, Casagrande G, Virador VM (2013) Mol Carcinog 52:167–182.

4. Soldatow VY, Lecluyse EL, Griffith LG et al. (2013) Toxicol Res (Camb) 2:23–39.

5. Zschenker O, Streichert T, Hehlgans S et al. (2012) PLoS One 7:e34279.

6. Smalley KS, Lioni M, Herlyn M (2006) In Vitro Cell Dev Biol Anim 42:242–247.

7. Schwartz MA, Chen CS (2013) Science 339:402–404.

8. Chai YW, Lee EH, Gubbe JD et al. (2016) PLoS One 11:e0162853.

Acknowledgments: This art icle was contr ibuted by Beth Lindborg, Cait l in Johnson, Yi Wen Chai,

Eu Han Lee, and Scott Brush, BRTI Life Sciences, Two Harbors, Minnesota (brt i l i fesciences.com).

Images were acquired at the University of Minnesota-University Imaging Center (UMN-UIC) faci l i ty;

we greatly appreciate UMN-UIC member Grant Barthel’s assistance with confocal microscopy.

Detect cell proliferation in 3D culturesIn addition to its essential role in development, cell proliferation is an important marker of cancer

cells and can serve as a target of anti-cancer therapies. The Invitrogen™ Click-iT™ EdU Imaging

Kits provide a simple, efficient proliferation assay that detects DNA synthesis using fluorescence

microscopy. In the Click-iT EdU assay, a modified thymidine analog (5-ethynyl-2´-deoxyuridine, or

EdU) is introduced into cells, incorporated into newly synthesized DNA, and then labeled with a

brightly fluorescent Invitrogen™ Alexa Fluor™ dye in a fast, highly specific click reaction. We used

the Click-iT EdU Alexa Fluor 488 Imaging Kit (with no changes to the supplied protocol) with 3D

cell cultures that were created by embedding HeLa cells into Cell-Mate3D matrix, and found that

proliferating cells (which fluoresce green) were easily visualized in 4-day-old cultures (Figure 2).

Detect apoptosis in 3D culturesThe Invitrogen™ CellEvent™ Caspase-3/7 Green Detection Reagent enables quick and reliable

quantitation of apoptotic cells in culture. This nonfluorescent substrate is composed of the

4–amino acid peptide DEVD, which contains the caspase-3/7 recognition site, conjugated to a

nucleic acid–binding dye. In the presence of activated caspase-3 or -7, the substrate is cleaved,

freeing the dye to bind to DNA and producing a bright green-fluorescent signal indicative of

apoptosis. To test this reagent with 3D cell cultures, we embedded AU565 breast cancer cells

in CellMate-3D matrix and cultured the cells for 12 days. During this time, one sample was

left untreated and an equivalent sample was treated with 100 µM paclitaxel (generic name of

Taxol™ pharmaceutical) to induce apoptosis. Samples were then stained with 15 µM CellEvent

Caspase-3/7 reagent (3 times the recommended concentration). Green-fluorescent apoptotic

cells were clearly seen in the paclitaxel-treated sample by confocal microscopy (Figure 3).

Apply fluorescence protocols to your 3D experimentsAlthough some optimization was required when applying fluorescent probes to 3D cell

cultures growing in Cell-Mate3D matrix, we found that generally the reagents were able to

penetrate the complex environment and accurately report cell health parameters and enzyme

activity. Analysis of cell function within 3D cell cultures will greatly benefit from the plethora

of fluorescent cell function probes available. Explore fluorescence-based cell analysis at

thermofisher.com/cellanalysisbp75. ■

Figure 2. Detection of proliferating cells in the

Cell-Mate3D matrix using Click-iT EdU staining.

HeLa cells were embedded in the Cell-Mate3D

matrix and cultured for 4 days. Proliferating cells were

detected using the Invitrogen™ Click-iT™ EdU Alexa

Fluor™ 488 Imaging Kit (green, Cat. No. C10337).

Cells were counterstained with the Invitrogen™

NucBlue™ Fixed Cell ReadyProbes™ Reagent (blue,

Cat. No. R37606), and imaged using an inverted

confocal microscope at 60x magnification.

Figure 3. Detection of apoptotic cells in the

Cell-Mate3D matrix using CellEvent Caspase-3/7

Green reagent. AU565 breast cancer cells were

embedded in the Cell-Mate3D matrix and cultured for

12 days. During this time, cells were (A) left untreated

or (B) treated with 100 µM paclitaxel to induce apop-

tosis. Cells were then incubated with the Invitrogen™

CellEvent™ Caspase-3/7 Green Detection Reagent

(15 µM, Cat. No, C10423) for 30 min to label apoptotic

cells with green fluorescence, counterstained with the

Invitrogen™ NucBlue™ Live ReadyProbes™ Reagent

(blue, Cat. No. R37605), and imaged using an inverted

confocal microscope at 20x magnification.

Co-stimulatory checkpoint Co-inhibitory checkpoint

Ant

igen

-pre

sent

ing

cells

T ce

lls

Ant

igen

-pre

sent

ing

cells

T ce

lls

PD-L1 (B7-H1)

PD-L2

PD1

B7-H3 (CD276)

B7-H4 (VTCN1)

HVEM4-1BB L (CD137L)

OX40L (CD252)

GAL9

?

BTLA (CD272)

TIM3

?

MHC II

LAG3

PD-L1 or PD-L2?

CD28

ICOSL (B7RP1)

OX40 (CD134)

CD80 or CD86

ICOS (CD278)

4-1BB (CD137)

CD70CD27

PVR (CD155)

CD40L (CD154) CD40

IDO

CTLA4 (CD152)

CD80 or CD86

GITR (CD357) GITRL

CD30 CD30L (TNFSF8)

LIGHT (CD258)

DR3

CD96

Nectin-2 (CD112)

TL1A

VISTA (B7-H5)

?

?

DNAM1 (CD226)

B7-CD28 family TNF/TNFR superfamily

PVR (CD155)

TIGIT

Others

Nectin-2 (CD112)

Unknown

VISTA (B7-H5)


BIOPrOBEs 75 rEsEArCH WITH ANTIBODIEs

Harness immune checkpoints to combat tumorsImmune checkpoint antibodies for flow cytometry, IHC, and functional bioassays.

Figure 1. Multiple co-stimulatory and co-inhibitory receptor–ligand interactions between antigen-presenting

cells (APCs) and T cells. T cell receptors (TCRs) detect antigens on the surface of APCs in the form of

antigen-complexed major histocompatibility complexes (MHCs), and this antigen-specific recognition is necessary

but not sufficient for an effective T cell response. For T cell activation or suppression, T cells must recognize

their cognate antigens through TCRs and then respond to co-stimulatory (for activation) or co-inhibitory (for

suppression) receptor–ligand interactions, examples of which are shown in this schematic. One important family

of membrane-bound molecules that bind both co-stimulatory and co-inhibitory receptors is the B7-CD28 family

shown in purple boxes; all of the B7 family members and their known ligands belong to the immunoglobulin

superfamily. Another major category of signals arise from tumor necrosis factor (TNF) family members (shown in

green boxes), which regulate the activation of T cells in response to cytokines.

Figure 2. The blockade of immune checkpoint PD-1/PD-L1 in immunotherapy. This figure shows PD-1 and

PD-L1 for illustrative purposes, although the concept likely applies to multiple immune checkpoints. (A) When

PD-1 on the T cell surface binds its ligand PD-L1 on the tumor cell, the T cell becomes inactivated, leaving the

tumor cell intact and growing. Blocking the activity of PD-1 or PD-L1 with inhibitors (e.g., antibodies) can prevent

the interaction of PD-1 and PD-L1, enabling the T cell to stay active and launch an anti-tumor immune response

to the tumor cell, releasing inflammatory signals such as IFN-gamma. (B) Immunohistochemical staining of human

brain tissue was performed using an anti-PD1 (CD279) polyclonal antibody (Cat. No. PA5-20350, 25 µg/mL).

(C, D) Unstimulated or PHA-stimulated normal human peripheral blood cells have been stained with PD-1 or

PD-L1 antibody as follows, and viable cells in the lymphocyte gate were detected using Invitrogen™ eBioscience™

Fixable Viability Dye eFluor™ 520 (Cat. No. 65-0867-14). PD-1 or PD-L1 antibodies include: (C) APC mouse IgG1

kappa isotype control (blue histogram, Cat. No. 17-4714-82) or APC anti–human CD279 (PD-1) (purple histogram,

Cat. No. 17-9969-42). (D) APC mouse IgG1 kappa isotype control (blue histogram) or APC anti–human CD274

(PD-L1, B7-H1) (purple histogram, Cat. No. 17-5983-42).

PD-1PD-L1

Anti–PD-L1

Anti–PD-1

T cells inhibited and tumor growing T cells activated and tumor dying

PD-1PD-L1

IFN-gamma etc.

A

Cou

nts

CStimulatedUnstimulated

CD279 (PD-1) APC CD279 (PD-1) APC

Cou

nts

DStimulatedUnstimulated

CD274 (PD-L1) APC CD274 (PD-L1) APC

B

As critical members of the adaptive immune

system, T cells are capable of mounting an

efficient immune response against tumor

cells. As T cells circulate in the body, they

are constantly interrogating proteins on the

surface of cells they encounter. If a cell is

identified as “foreign”, the T cells will unleash

an attack against the invading cell. Tumor

cells, however, can evade detection by

masking their surface with proteins of normal

cells. These proteins—referred to as immune

checkpoints—can affect immuno regulatory

pathways by either boosting (co-stimulatory)

or restricting (co-inhibitory) the immune

responses of T cells [1,2]. So far, a great

number of immune checkpoint proteins have

been identified (Figure 1).

Antibodies for dissecting the immune checkpoint pathwaysAntibodies that target immune checkpoint

pathways—such as PD-1/PD-L1 (Figure 2),

CTLA4/CD80 and CD86, LAG3/MHC II,

and TIM3/GAL9—have proven effective

as immuno therapeutic agents in cancer

treatment. For example, PD-1/PD-L1 is a

co-inhibitory pathway that functions to restrict

the immune system by constraining T cell

anti-tumor activity. Studies with antibodies

(e.g., nivolumab and pembrolizumab) that

target the PD-1/PD-L1 pathway and suppress

its co-inhibitory function have shown durable

clinical responses in combating certain can-

cers, even in patients with advanced-stage

cancer [1,2].

Recently, the co-inhibitory receptor

TIGIT has been found to be expressed on

the surface of a variety of lymphocytes,

CD45RO PerCP-eFluor 710

TIG

IT P

E

CD45RO PerCP-eFluor 710

Mou

se Ig

G1

PE


rEsEArCH WITH ANTIBODIEs BIOPrOBEs 75

References1. Topalian SL, Drake CG, Pardoll DM (2015) Cancer Cell 27:450–461.

2. Pardoll DM (2012) Nat Rev Cancer 12:252–264.

3. Kurtulus S, Sakuishi K, Ngiow SF et al. (2015) J Clin Invest 125:4053–4062.

4. Nishikii H, Kim BS, Yokoyama Y et al. (2016) Blood 128:2846–2858.

Figure 3. Expression of TIGIT on CD4+ CD45RO+

T cells. Normal human peripheral blood cells were

stained with APC anti–human CD4 antibody (clone

RPA-T4, Cat. No. 17-0049-42), PerCP-eFluor™ 710

anti–human CD45RO antibody (clone UCHL1, Cat.

No. 46-0457-42), and PE mouse IgG1 kappa isotype

control (Cat. No. 12-4714-82) (top) or PE anti–human

TIGIT antibody (bottom, Cat. No. 12-9500-42). CD4+

cells in the lymphocyte gate were used for analysis.

Figure 4. Expression of DR3 in Ramos cells. Immunocytochemical fluorescence analysis was performed on

fixed and permeabilized Ramos cells for detection of (A) endogenous DR3 (TNFRSF25) using Invitrogen™ anti-DR3

(TNFRSF25) ABfinity™ recombinant rabbit monoclonal antibody (Cat. No. 702277, 2 µg/mL) in conjunction with

Invitrogen™ Alexa Fluor™ 488 goat anti–rabbit IgG Superclonal™ secondary antibody (green; Cat. No. A27034,

1:2000 dilution), (B) nuclei using Invitrogen™ SlowFade™ Gold Antifade Mountant with DAPI (blue, Cat. No.

S36938), and (C) cytoskeletal F-actin using rhodamine phalloidin (red, Cat. No. R415, 1:300 dilution). (D) The

composite image shows staining of DR3 (TNFRSF25), nuclei, and actin. To assess background fluorescence,

control cells were stained similarly except that no primary antibody was used (data not shown). The images were

captured at 60x magnification on a Nikon™ Eclipse™ Ti-U inverted microscope.

including effector and regulatory CD4+ T cells,

follicular helper CD4+ T cells, effector CD8+

T cells, natural killer (NK) cells, and memory

T cells (Figure 3). The expression of TIGIT is

markedly enriched on tumor-infiltrated T cells

[3]. In addition, the TIGIT+ regulatory T cells in

tumor tissue co-express TIM3, and TIM3 and

TIGIT work together to suppress anti-tumor

immunity [3]. Similarly, therapeutic agonists

of DR3, a co-stimulatory receptor that is

expressed primarily in tissues enriched in

lymphocytes (Figure 4), can also be used to

stimulate regulatory T cell expansion, which

can reduce inflammation and enable patients

to produce an effective anti-tumor response

[4]. These examples show that releasing the

anti-tumor immune response by blocking

co-inhibitory immune checkpoints or activating co-stimulatory immune checkpoints is a prom-

ising approach for triggering anti-tumor responses and mediating durable cancer regressions.

Check out our checkpoint antibodiesDespite recent breakthroughs in immunotherapy to treat melanoma of the skin, non-small cell

lung cancer, kidney and bladder cancers, head and neck cancers, and Hodgkin lymphoma, the

complex biology of the immune checkpoint pathways is still far from being understood. We offer

a wide range of antibodies designed to study immune checkpoints (Table 1), as well as blocking

antibodies, proteins, assays, and more. Our experienced custom service team can also save you

time by developing antibodies to meet your specifications; visit thermofisher.com/antibodies

and select “Custom Antibodies” to learn more. ■



Table 1. Selected antibodies for flow cytometry, immunohistochemistry (IHC)/immunofluorescence (IF), and functional bioassays.

Immune checkpoint

Anti-human antibody (Cat. No.) Anti-mouse antibody (Cat. No.)

Flow cytometry IHC/IFFunctional bioassay Flow cytometry IHC/IF

Functional bioassay

B7-H3 (CD276) MA1-74441, 46-2769-42 MA5-15693, MA1-74441 16-5937-85 16-5973-81 16-5973-81

B7-H4 (VTCN1) MA5-16845, 17-5949-42 MA1-74439, MA1-74440 16-5949-82 12-5970-83, 12-5972-82 16-5972-81

B7-H5 (VISTA) PA5-52493 46-5919-82 PA5-47032 16-5919-85

BTLA (CD272) MA5-16843, 13-5979-80 PA5-22248, MA1-74212 16-5979-82 12-5950-82, 17-5956-82 16-5950-82

CD27 11-0279-42, 11-0271-85 16-0272-85, PA5-28036 16-0271-85 MA5-17902, MA5-17904 12-0271-83 16-0272-85

CD28 61-0289-42, MA1-10170 MA1-10166, MA5-17005 16-0281-86 MA1-34061, 14-0281-86 MA1-10172 16-0289-85

CD30 MA5-12632, 11-0309-42 MA5-12632 12-0301-82 MA1-90234 16-0301-85

CD30L (CD153) MA5-23859 PA5-47045 MA5-23859 A18356, 12-1531-83 14-1531-85 14-1531-85

CD40 MA1-80926, 11-0409-42 14-0409-82, 700121 16-0409-85 MA5-17853, 46-0401-82 14-0401-86 16-0402-86

CD40L (CD154) MA1-33895, 11-1548-42 PA5-13483 16-1548-82 HMCD15401, 46-1541-82 MA1-10108 16-1541-85

CD70 MA5-17727, 50-0709-42 PA5-32701, MA5-17726 14-0701-85, MA1-81645 16-0701-85

CD80 MA1-19215, 11-0809-42 MA5-15512, MA1-90872 16-0809-85 MA1-70090, 11-0801-86 14-0801-85 16-0801-85

CD86 MA1-81186, 12-0869-42 MA1-12172, MA1-10293 14-0869-82 12-0861-83, 11-0862-85 14-0862-85 16-0861-85

CD96 (Tactile) 46-0969-42, MA5-24280 PA5-64286 MA5-24281, 12-0960-80 16-0960-82

CD112 (Nectin-2) MA1-35890, 17-1128-42 PA5-29757

CD134 (OX40) MA5-23591, 11-1347-42 PA5-34516, 14-1347-82 12-1341-83, MA1-70087 14-1341-85, MA5-17916 16-1341-85

CD137 (4-1BB) MA5-13739, 11-1379-42 MA5-13739 PA5-47037 PA5-47967, 25-1371-82 16-1371-85

CD137L (4-1BB L) PA5-47291 46-5901-82

CD152 (CTLA-4) 12-1528-42, 25-1529-42 PA5-23967, PA5-47547 16-1529-82 61-1522-82, HMCD15201 16-1521-85

CD155 (PVR) MA5-13493, 46-1550-42 MA5-13490 17-1551-82 MA5-24315

CD223 (LAG3) 46-2239-42 MA5-24284, 11-2231-82 16-2231-85

CD226 (DNAM1) PA5-38444, PA5-31111 MA5-17990, 16-2261-85 PA5-36393 16-2261-85

CD252 (OX40L) PA5-24552 PA5-34516 46-5905-82

CD258 (LIGHT) 46-2589-42, 17-2589-42

CD273 (PD-L2) 17-5888-42, MA5-16839 PA5-20344 16-5888-82 12-5986-83, 11-9972-85 14-5986-85 16-5986-82

CD274 (PD-L1) 46-5983-42, MA5-16840 14-5983-82 16-5983-82 62-5982-80, 13-9971-81 14-5982-85 16-5982-85

CD278 (ICOS) 11-9948-42, MA5-23680 14-9948-82 16-9948-82 46-9940-82, 11-9942-82 14-9949-82 16-9942-85

CD279 (PD-1) 12-9969-42, 12-2799-42 14-2799-80, 14-9969-82 16-9989-38 11-9981-82, 25-9985-82 14-9985-85 16-9985-85

CD357 (GITR) 48-5875-42, MA5-23854 PA5-46810, PA5-47883 PA5-47883 MA5-17934, 11-5874-82 16-5874-83 16-5874-83

DR3 (TNFRSF25) 702277, 12-6603-42 PA1-30533, PA5-28293 711309, MA5-23838 711309

Galectin-9 50-9116-42 PA5-32252, PA5-50966 16-9116-85 PA5-47503 MA5-24369

GITRL PA5-20161 MA5-23852, PA5-47885

PA5-20161

HVEM MA1-25958, 12-5969-80 PA5-20236, PA5-26103 17-5962-82 PA5-20237 16-5962-85

ICOSL (B7RP1, B7-H2)

MA1-17756, 12-5889-42 13-5889-82 16-5889-82 50-5985-82, MA5-24270 PA5-47161 16-5985-85

IDO MA5-23595, 12-9477-42 PA5-29819, PA5-12305 46-9473-82 PA5-24598

TIGIT 46-9500-42 16-9500-85 12-9501-82 16-9501-85

TIM3 47-3109-42 16-3109-85 16-5871-85, MA5-1795 14-5871-85 16-5871-85

TL1A 46-7911-82 46-7911-82

VSIG4 17-5757-42 PA5-52018 17-5752-82



Comprehensive strategy for antibody validationCharacterization of antibody performance using immunoprecipitation and mass spectrometry.

Antibodies serve as essential tools in many facets of biological

research. They are used as sensitive detection reagents to interrogate

cell pathways, to diagnose disease and assess different treatment

strategies, and to effect immunotherapeutic responses of their own.

Poorly characterized antibodies not only waste time and money but

also can lead to flawed research findings and incorrect conclusions [1].

To address these issues, several funding agencies and major

journals will begin requiring additional antibody information in 2017,

and many antibody suppliers and research labs are unprepared for

these changes. Providing guidance to the research community, the

International Working Group on Antibody Validation (IWGAV) published

a Nature Methods manuscript in September 2016 that described five

recommended “conceptual pillars” to guide antibody validation [2].

These include:

■ Genetic strategies: Measure the relevant signal in control cells or

tissues in which the target gene has been knocked out or knocked

down using techniques such as CRISPR-Cas or RNAi.

■ Orthogonal strategies: Use an antibody-independent method

for quantitation across multiple samples, and then examine the

correlation between the antibody-based and antibody-independent

quantitations.

■ Independent antibody strategies: Use two or more independent

antibodies that recognize different epitopes on the target protein

and confirm specificity with comparative and quantitative analyses.

■ Expression of tagged proteins: Modify the endogenous target

gene to add sequences for an affinity tag or a fluorescent protein.

The signal from the tagged protein can then be correlated with

antibody-based detection.

■ Immunocapture followed by mass spectrometry (IP-MS):

Couple immunocapture (i.e., immunoprecipitation, or IP), the tech-

nique of isolating a protein from a solution through binding with a

target-specific antibody, with mass spectrometry (MS) analysis to

identify proteins that interact directly with the purified antibody as

well as proteins that may form a complex with the target protein.

While each of these conceptual pillars may provide evidence of anti-

body specificity, the IWGAV recommends multiple pillars be used in

order to claim a particular antibody has been well validated for use in

a specific application. The IWGAV paper has examples of the first four

conceptual pillars; however, the paper shows no data for the IP-MS

strategy, which is the only validation approach that can verify the true

Figure 1. Experimental workflow for antibody valida-

tion by immunoprecipitation and mass spectrometry

(IP-MS) analysis. Protein targets and antibodies are

prioritized for validation based on research areas and

literature references. Cell models are then identified

based on literature references and RNA expression,

and lysates from these cell lines are prepared for MS

analysis by cysteine reduction and alkylation, tryptic

digestion, high-pH reversed-phase fractionation, and

peptide quantitation. Fractionated peptide samples are

analyzed by nanoLC-MS/MS on a Thermo Scientific™

Q Exactive™ Mass Spectrometer, and peptides are iden-

tified and quantified with Thermo Scientific™ Proteome

Discoverer™ 2.1 and MaxQuant software (v1.5.8, Max

Planck Institute). Protein targets are immuno-enriched

from cell lysates with the Thermo Scientific™ Pierce™

MS-Compatible Magnetic IP Kit (protein A/G) and

analyzed by nanoLC-MS/MS to verify and quantify

target fold-enrichment. After filtering to remove common

background proteins, enriched proteins are submitted

for analysis of known interactions with the STRING

database (http://string-db.org). These antibodies can be

used alone or in combination for enrichment of protein

targets prior to MS-based quantitation.

Cell line 1Cell line 2Cell line 3Cell line 4

Cell line N

Pro

tein

1P

rote

in 2

Pro

tein

3P

rote

in 4

Pro

tein

N

Select targetsand antibodies

Select cell models,grow cells, make lysates

Immuno-enrichtargets

Bioinformatic analysis

MS analysis of cell models

Prepare samplesand analyze by MS

TUBA1C

CTNNA1

JUP MYO6DCD

CDH1

MRPS12

RPL38

CTNNB1

THADAKRT16ABHD11

LAD1 MYO5B NCOA3

MYO5AKCMF1

CTNNA2

57 35

26 24

20 20

14 13

11 11

6 6

4 4

3 2 2 2 2

0 10 20 30 40 50 60

CDH1 CTNNA1 NCOA3 MYO6

CTNNB1 THADA

JUP FAM127A/B/C

CTNNA2 DCD

KCMF1 MYO5A KRT16

ABHD11 RPL38

LAD1 MRPS12 TUBA1C MYO5B

Fold enrichment

Targeted protein

Direct CDH1 interactor



antibody target as well as identify protein modifications, isoforms,

off-target(s), and interacting proteins. In order to better characterize

Invitrogen™ antibodies, we have implemented a comprehensive

workflow for antibody validation * using immunocapture followed by

mass spectrometry (Figure 1). This strategy includes the selection of

protein targets, antibody candidates, and cell models, as well as the

characterization of cell models by LC-MS, IP-MS sample preparation

and analysis, and bioinformatic analysis.

Selecting protein targets, antibody candidates, and cell linesWe choose protein targets based on research areas (e.g., cancer

signaling, stem cell differentiation, etc.) and literature references.

We then identify candidate cell lines likely to express these proteins

based on RNA expression, followed by extensive characterization of

the proteomes of those cell lines with liquid chromatography in-line

with mass spectrometry (LC-MS). The LC-MS data verify the presence

of the target protein in the cell line and provides protein quantitation

information. For our initial round of antibody validation, we chose 12 cell

lines from the NCI60 cancer cell line panel based on RNA expression

data, and identified and quantified 6,000–9,000 proteins from each cell

line by LC-MS. For each antibody candidate, we use this information

to choose cell lines that express the antibody’s target protein at a mid-

to-low expression level so that we can test the antibody performance

in a relevant background. We then immunoprecipitate (IP) the protein

target from the cell lysate with each antibody candidate and analyze the

enriched proteins by LC-MS. Finally, we verify that the target is detected,

and we conduct a bioinformatic analysis of the IP-MS data (Figure 2).

Assessing antibody selectivity and identifying interacting proteins with IP-MSMS can be used to detect every protein in an immunoprecipitated

sample. However, unlike western blotting or ELISA techniques, MS

is not compatible with the use of a blocker, such as milk or albumin,

to minimize nonspecific binding. Instead, we can filter out common

contaminating proteins from the LC-MS analysis by comparing the

immunoprecipitated proteins and their abundance with proteins immu-

noprecipitated by an antibody to an unrelated protein target, which

serves as a negative control. This comparison simplifies the detection

of the target protein, as well as any off-targets and interacting proteins.

After subtracting background proteins from the IP-MS data using a

negative control, we calculate the fold enrichment of identified proteins

after immunoprecipitation relative to the protein expression level in the

cell line tested, using the formula:

Figure 2. Selection of cell lines for antibody verification by IP-MS. (A) RNA expression Z-scores from the NCI60 cell line panel were retrieved from Cell Miner

(https://discover.nci.nih.gov/cellminer/) and hierarchically clustered for 22 genes in the Thermo Scientific™ Ion AmpliSeq™ Colon and Lung Cancer Panel. (B) Venn diagram

of the number of proteins identified from five NCI60 cell lines using mass spectrometry analysis of peptides from each lysate.

BRAF

CTNNB1

FBXO7

AKT1

DDR2

PIK3CA

ERBB2

ERBB4

FGFR2

FGFR3

ALK

NOTCH1

KRAS

PTEN

TP53

EGFR

MET

MAP2K1

STK11

FGFR1

SMAD4

NRAS LE:C

CR

F_CE

M

LE:M

OLT_4

LE:H

L_60

LE:K

_562

LE:R

PM

I_8226

CO

:HC

C_2998

CO

:HT29

CO

:KM

12

CO

:CO

LO205

LC:N

CI_H

322M

BR

:MC

F7

CN

S:U

251

BR

:MD

A_M

B_231

CN

S:S

NB

_19

ME

:LOX

IMV

I

CO

:SW

_620

LC:N

CI_H

23

LC:N

CI_H

460

PR

:DU

_145

ME

:MA

LME

_3M

ME

:SK

_ME

L_5

ME

:UA

CC

_257

ME

:SK

_ME

L_2

ME

:M14

ME

:SK

_ME

L_28

ME

:UA

CC

_62

ME

:MD

A_M

B_435

ME

:MD

A_N

CN

S:S

F_539

LC:H

OP

_62

PR

:PC

_3

BR

:T47D

OV

:IGR

OV

1

OV

:OV

CA

R_3

OV

:OV

CA

R_4

LC:N

CI_H

522

LE:S

R

BR

:BT_549

CN

S:S

NB

_75

CN

S:S

F_268

LC:N

CI_H

226

BR

:HS

578T

CN

S:S

F_295

LC:H

OP

_92

RE

:SN

12C

RE

:UO

_31

CO

:HC

T_15

CO

:HC

T_116

RE

:CA

KI_1

OV

:OV

CA

R_8

OV

:NC

I_AD

R_R

ES

RE

:786_0

RE

:TK_10

LC:E

KV

X

RE

:RX

F_393

LC:A

549

RE

:A498

RE

:AC

HN

OV

:OV

CA

R_5

OV

:SK

_OV

_3

1,305

226

256

180

342

191357

246

185

129

345

3,611248

345229

109

1,014

1,273

908

1,580

306

173

132

197

281

136

155

275

275 159191

HepG

2

A549

BT549

MCF7

LNCaP

BA

Target protein abundance in IP sample

Total protein abundance in IP sampleFold enrichment =

Target protein abundance in cell lysate

Total protein abundance in cell lysate



References1. Bradbury A, Plückthun A (2015) Nature 518:27–29.

2. Uhlen M, Bandrowski A, Carr S et al. (2016) Nat Methods 13:823–827.

Figure 3. Fold enrichment of cadherin-1 (CDH1) and interacting proteins. (A) CDH1 was enriched >50-fold from MCF7 cell lysate using anti-CDH1 monoclonal antibody.

Identified proteins were submitted to STRING (http://string-db.org) for interactome analysis. Based on the STRING results, we color-coded the fold-enrichment bars for

CDH1 (red) and its likely interactors (blue). (B) STRING interactome diagram for proteins enriched with an anti-CDH1 antibody.

Fold enrichment is a measure of the ability of an antibody to

selectively capture its target protein. The calculated fold enrichment

of each protein captured with an antibody is plotted to assess the

capture performance and selectivity of the antibody (Figure 3A). This

list of enriched proteins is then analyzed with the STRING database

(http://string-db.org), a public repository of known or predicted protein

interactions; the STRING database website can provide a diagram

of known or suspected protein interactions from the list of immuno-

precipitated proteins (Figure 3B). We then color-code the fold-enrichment

bar chart to signify the intended target (red) and any observed likely

interactors (blue) from the STRING analysis. For example, cadherin-1

(CDH1, E-cadherin) is enriched with an anti–cadherin-1 antibody, as

are several proteins that are known to interact with CDH1 including

α- and β-catenin (Figure 3), and these results are observed with several

standard cell lines. In other experiments, antibodies to β-catenin also

enrich CDH1 (data not shown). Several other common background

proteins that may stick to tubes and beads, such as myosin (MYO6),

are also present, suggesting that this IP-MS analysis does not over-filter

common background proteins.

Learn more about our IP-MS validated antibodiesIP-MS can be used to verify the antibody target and to identify

off-targets, interacting proteins, and protein modifications. This

in-depth antibody characterization approach provides verification

data that are not available with any other validation technique, and

it is only offered by Thermo Fisher Scientific. IP-MS validated anti-

bodies can be used for simultaneous enrichment of low-abundance

signaling pathway proteins, as described in “Examine signaling

pathways with targeted proteomics” on page 19. For more details

on this validation approach or our most recent validation data, visit

thermofisher.com/antibodyvalidationbp75. ■

57 35

26 24

20 20

14 13

11 11

6 6

4 4

3 2 2 2 2

0 10 20 30 40 50 60

CDH1 CTNNA1 NCOA3 MYO6

CTNNB1 THADA

JUP FAM127A/B/C

CTNNA2 DCD

KCMF1 MYO5A KRT16

ABHD11 RPL38

LAD1 MRPS12 TUBA1C MYO5B

Fold enrichment

Targeted protein

Direct CDH1 interactor

TUBA1C

CTNNA1

JUP MYO6DCD

CDH1

MRPS12

RPL38

CTNNB1

THADAKRT16ABHD11

LAD1 MYO5B NCOA3

MYO5AKCMF1

CTNNA2


Pierce™ BCA Protein Assay Kit 1 L 23225

Pierce™ High pH Reversed-Phase Peptide Fractionation Kit 12 reactions 84868

Pierce™ IP Lysis Buffer 250 mL 87788

Pierce™ Mass Spec Sample Prep Kit for Cultured Cells 20 reactions 84840

Pierce™ MS-Compatible Magnetic IP Kit, protein A/G 40 reactions 90409

Pierce™ Quantitative Colorimetric Peptide Assay 500 assays 23275

Pierce™ Trypsin Protease, MS Grade 5 x 20 µg 90057

* The use or any variation of the word “validation” refers only to research use

antibodies that were subject to functional testing to confirm that the antibody

can be used with the research techniques indicated. It does not ensure that

the product(s) was validated for clinical or diagnostic uses.

BA



Examine signaling pathways with targeted proteomicsQuantitative analysis of the AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry.

The AKT/mTOR pathway plays a central

role in tumor progression and cancer-drug

resistance, and therefore the quantitative

measurement of this pathway’s protein

expression and posttranslational modifica-

tions (PTMs) is vital to cancer research [1]. A

major limitation when measuring AKT/mTOR

pathway protein levels is the lack of rigor-

ously validated methods and reagents, as

well as a reliance on semiquantitative results

from western blot analyses. Because many

biologically relevant proteins are present in

vanishingly small quantities, immunoprecipi-

tation (IP) is commonly used as a tool for

enriching protein [2,3]. Mass spectrometry

(MS) is increasingly becoming the detection

method of choice for determining protein

abundance and identifying PTMs.

The IP-MS workflow, which combines IP

steps with subsequent analysis by MS, can be

used to enrich signaling proteins, benchmark

antibody performance, and reveal protein–

protein interactions [4]; see “Comprehensive

strategy for antibody validation” on page 16 for more information. Multiplex IP coupled with

targeted MS (mIP-tMS) further enhances this workflow by simultaneously quantifying multiple

proteins and their phosphorylation states in a specific signaling pathway. Here we demonstrate

the mIP-tMS methodology by analyzing a specific set of protein targets in the AKT/mTOR pathway.

Singleplex IP-MS assay development for AKT/mTOR pathway proteinsTo begin our analysis of the AKT/mTOR pathway, we selected 11 pathway proteins—10 of

which have at least one phosphorylation site—for antibody validation by IP-MS (Table 1). These

targets were chosen to align with commercially available reagents for western blot analysis (WB),

enzyme-linked immunosorbent assays (ELISA), and Luminex® bead-based multiplex immuno-

assays (Luminex assays). Figure 1 shows the general workflow for mIP-tMS assay development.

The first step was to validate antibodies by IP-MS and identify quantotypic peptides for each

AKT/mTOR pathway protein. Antibody candidates were screened by IP-MS to determine their

effectiveness—both their ability to immunoprecipitate AKT pathway proteins and their usefulness

when combined with MS. The IP-enriched samples were then analyzed by LC-MS to qualitatively

identify targets of interest, interacting proteins, PTMs, and quantotypic peptides. We performed

the LC-MS analysis using a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano System

and Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer, and

the data were analyzed with Thermo Scientific™ Proteome Discoverer™ 1.4 software to

Figure 1. mIP-tMS assay workflow. Multiple antibodies for the 11 total and 10 phosphorylated AKT/mTOR

pathway protein targets were validated using the Thermo Scientific™ Pierce™ MS-Compatible Magnetic IP Kit

(protein A/G) and analyzed by LC-MS. The antibody with greatest capture efficiency for each target was then

selected for biotinylation using the Thermo Scientific™ Pierce™ Antibody Biotinylation Kit for IP. Lastly, the 11 total

and 10 phosphorylated protein targets were simultaneously enriched by mIP using validated biotinylated anti-

bodies and the Thermo Scientific™ Pierce™ MS-Compatible Magnetic IP Kit (streptavidin). mIP eluate samples

were processed by an in-solution digestion method to generate a proteotypic peptides mix. Internal standard

heavy peptide mix for 11 AKT/mTOR pathway proteins was spiked into digested peptides, and tMS assay was

performed in parallel reaction monitoring (PRM) mode for quantitation of 11 pathway proteins in a single MS run.

Select total andphospho targets

Grow cells andstimulate with IGF

LC-MS analysis

Immuno-enrichtargets (protein A/G)

Identify quantotypicpeptides and

develop tMS assays

Antigens

Antibody

Time (min)

Multiplexed PRM/SRMquanti�cation

Multiplexed proteinimmunocapture (streptavidin)

Biotin

Table 1. Total and phosphorylated AKT/mTOR pathway protein targets selected for analysis.

Protein target Phosphorylation site

AKT1/AKT2 pSer473/pSer474

mTOR pSer2448

IGF1R pTyr1135/1136

IR NA

PRAS40 pThr246

p70S6K pThr389

TSC2 pSer939

PTEN pSer380

GSK3α pSer21

GSK3β pSer9

IRS1 pSer312



assess percent sequence coverage, unique peptides, area, and PTMs

for each pathway protein.

We identified the unique tryptic peptides with the best analytical

characteristics for each protein target, and selected these quantotypic

peptide sequences for the synthesis of stable isotope–labeled AQUA

peptides (using HeavyPeptide™ AQUA Ultimate custom services) that

serve as internal standards. Heavy AQUA peptides for each protein

target were further evaluated for linearity, reproducibility, accuracy,

dynamic range, quantitation limits, and recovery.

Finally, absolute quantitation of each protein target was achieved

by utilizing a standard curve with serial dilutions of each heavy AQUA

peptide across three orders of magnitude, in conjunction with targeted

MS (tMS) using the parallel reaction monitoring (PRM) method imple-

mented on the mass spectrometer. The tMS data were analyzed with

Skyline software (MacCoss Lab Software, University of Washington) to

determine the limit of quantitation (LOQ) from the calibration curve and

the target analyte concentration from unknown samples.

Enrichment of AKT/mTOR pathway protein targetsTable 2 shows the enrichment of low-abundance AKT/mTOR path-

way protein targets by the singleplex IP-MS method. IGF stimulation

has been shown to activate AKT/mTOR pathway signaling through

phosphorylation cascades [5]. AKT/mTOR pathway protein targets

were immunoprecipitated from unstimulated and IGF-stimulated A549

lysates for MS analysis using the IP-MS methodology described above.

When compared with neat lysates (combined lysates from the unstim-

ulated and IGF-stimulated cells that were not immunoprecipitated),

the IP-enriched samples and LC-MS analysis allowed us to identify a

significantly larger number of unique peptides (Table 2).

The effect of IGF stimulation on AKT/mTOR pathway proteins

was assessed by comparing the abundance of signaling proteins,

interacting partners, and PTMs for each protein target. Protein iso-

forms and interacting partners were identified for total AKT, IGF1R,

and mTOR targets. Relevant phosphorylation sites were detected for

phosphorylated AKT1, AKT2, mTOR, IGF1R, and PRAS40 targets.

Relative abundance of each pathway protein was determined using

the total area of all unique peptides identified for each target. A larger

number of unique peptides for phosphorylated AKT and IGF1R were

observed for IGF-stimulated A549 lysates compared with unstimulated

samples. These results demonstrate that beyond simply identifying

protein targets from whole lysates, the singleplex IP-MS method can

Table 2. Enrichment of low-abundance AKT/mTOR pathway proteins by IP-MS.

IP antibody

Targets identified

Neat no. of unique peptides

IP-enriched no. of unique peptides Relevant

phospho-peptide ID–IGF +IGF

Phospho-AKT

AKT1 – 3 20 +IGF: Ser473

AKT2 – – 14 +IGF: Ser474

AKT3 – – 13 NA

AKT1

AKT1 – 16 12 NA

AKT2 – 9 11 NA

AKT3 – 5 3 NA

Phospho-mTOR

mTOR 2 75 82Thr2446, Ser2448

RICTOR – – 2 NA

SIN1 – 2 3 NA

Gbl – 4 4 NA

IGF1RIGF1R 4 13 13 NA

IR – 10 6 NA

Phospho-IGF1R

IGF1R 4 – 5+IGF:

Tyr1135/1136

PRAS40 PRAS40 – 8 8 Thr246

Phospho-PRAS40

PRAS40 – 8 6 Thr246

also be applied to interrogate AKT/mTOR pathway signaling events in

the context of IGF stimulation.

Validation of mIP-tMS assaysThe singleplex IP-MS analysis described above highlighted antibodies

that successfully enrich their intended targets, interacting proteins, and

PTMs; however, it required that we perform separate IPs for each of the

10 phosphorylated (and 11 total) protein targets from the AKT/mTOR

pathway. Alternatively, mIP-tMS analysis should allow simultaneous

enrichment and quantitation of all targets from a single IP. Focusing on

the AKT/mTOR proteins that we identified earlier, we validated the use

of 10-plex phospho and 11-plex total mIP-tMS assays with unstimulated

and IGF-stimulated MCF7 lysates. mIP was carried out using biotinylated

antibodies and the Thermo Scientific™ Pierce™ MS-Compatible Magnetic

IP Kit (streptavidin). For the tMS analysis, standard curves were gener-

ated using 18 spiked-in AQUA heavy peptides for 12 targets (including

both AKT1 and AKT2) and then employed for absolute quantitation of

AKT/mTOR proteins. These mIP-tMS assays identified 11 proteins in the

multiplex phospho assay and 12 proteins for the multiplex total assay

(Table 3). We found that in addition to the IRS1 protein, PI3K subunits

were identified in IGF-stimulated MCF7 cell lysates. Upregulation of phos-

phorylated AKT1, AKT2, and IGF1R was observed upon IGF stimulation.




Pierce™ MS-Compatible Magnetic IP Kit, protein A/G 40 reactions 90409

Pierce™ Antibody Biotinylation Kit for IP 8 reactions 90407

Pierce™ MS-Compatible Magnetic IP Kit, streptavidin 40 reactions 90408

PEPotec SRM Custom Peptide Libraries Custom

HeavyPeptide™ AQUA-Grade Standards Custom

References1. Logue JS, Morrison DK (2012) Genes Dev 26:641–650.

2. Gingras AC, Gstaiger M, Raught B et al. (2007) Nat Rev Mol Cell Biol 8:645–654.

3. Carr SA, Abbatiello SE, Ackermann BL et al. (2014) Mol Cell Proteomics 13:907–917.

4. Ackermann BL (2012) Clin Chem 58:1620–1622.

5. Kodama Y, Baxter RC, Martin JL (2002) Am J Respir Cell Mol Biol 27:336–344.

Figure 2. Technology benchmarking of the AKT/mTOR pathway. Comparison of mIP-tMS assays with current immunoassay techniques to quantitate AKT-mTOR path-

way protein targets from unstimulated and IGF-stimulated A549, HCT116, and MCF7 lysates were performed. Western blot, ELISA, and Luminex assays were performed

according to manufacturer’s instructions. mIP-tMS assays for 11 total and 10 phosphorylated targets were performed in PRM mode using the Thermo Scientific™ Q Exactive™

HF Hybrid Quadrupole-Orbitrap Mass Spectrometer. Good correlation was observed for (A) total AKT and (B) phospho IGF1R for mIP-tMS assays, ELISA, and Luminex

assays but not western blot. RQ = relative quantitation; AQ = absolute quantitation.

0

1

2

3

2.6

2.7

2.8

2.9

3.0

Fluo

resc

ence

inte

nsity

(x 1

04 )

Ab

sorb

ance

(450

nm

)(-IGF)(+IGF)

BA

HCT116 A549 MCF7 HCT116 A549 MCF7

LuminexRQ RQ ELISA

HCT116 A549 MCF7 HCT116 A549 MCF7

Western blotRQ AQ tMS (PRM)

0

2

4

6

8

10

12

14

Vol

ume

inte

nsity

(x 1

06 )

0

10

20

30

40

50

Con

cent

ratio

n (fm

ol/I

P)

HCT116 A549 MCF7

Luminex

HCT116 A549 MCF7

ELISA

0

20

40

60

80

Con

cent

ratio

n (n

g/m

L)

0

0.4

0.8

1.2

1.6

2.0

Ab

sorb

ance

(450

nm

)

HCT116 A549 MCF7

Western blot

HCT116 A549 MCF7

tMS (PRM)

0

4

8

12

16

Con

cent

ratio

n (fm

ol/I

P)

0

2

4

6

AQ

AQ

RQ

RQ

Vol

ume

inte

nsity

(x 1

06 )

Table 3. Validation of phospho and total mIP-tMS assays.

Target

10-plex phospho assay 11-plex total assay

Number of unique peptides

–IGF +IGF –IGF +IGF

AKT1 – 9 25 30

AKT2 – 4 24 26

mTOR 48 56 25 28

IGF1R 1 3 32 35

IR NA NA 29 26

PRAS40 5 7 9 10

p70S6K 9 14 11 12

TSC2 5 10 42 45

PTEN – 1 5 9

GSK3α 7 6 19 21

GSK3β 13 10 23 23

IRS1 4 11 45 54

PIK3R1 – – – 22

PIK3CA – – – 2

PIK3CB – – – 6

PIK3R2 – – – 22

Benchmarking mIP-tMS assaysSeveral singleplex or multiplex immunoassay-based techniques exist for

quantifying proteins of interest from biological samples. We sought to

compare the mIP-tMS assay with established immunoassays in order

to benchmark its analytical performance. We analyzed 11 phosphory-

lated and 12 total AKT/mTOR pathway proteins in unstimulated and

IGF-stimulated HCT116, A549, and MCF7 cells using mIP-tMS, WB,

ELISA, and Luminex assays (Figure 2). mIP-tMS assays allowed absolute

quantitation for all 12 total and 11 phosphorylated targets in low- to

subnanogram concentrations across all cell lines. The comparative

summary of the four techniques showed target-dependent correla-

tion, and the WB method disagreed most often with the other three

methods (Figure 2). Variability across techniques for some targets can

be the result of antibody specificity. Phosphorylated targets showed

lower correlation compared to total protein abundances from the

AKT/mTOR pathway.

ConclusionsUsing the mIP-tMS method developed for the AKT/mTOR pathway, we

verified antibody selectivity and assessed interactions and off-targets

for AKT/mTOR pathway proteins. Validated antibodies and optimized

mIP-tMS workflows combine the benefits of target enrichment, selec-

tivity, and flexibility to better interrogate complex biological interactions.

The multiplexing capabilities of mIP-tMS assays provide an effective

strategy for increasing sample throughput without sacrificing detection

limits. For more information on our suite of tools for mass spectrometry,

visit thermofisher.com/massspecbp75. ■


TOOLs FOr FLOW CYTOMETrY BIOPrOBEs 75

Bright fluorescent polymer dyes for the violet laserSuper Bright antibody conjugates.

Flow cytometry is an important technique that is central to many

of the fastest-growing areas of life science research, including

immuno-oncology, antibody therapeutic development, and gene edit-

ing. Not only does it enable simultaneous multiparametric analysis of

proteins, gene expression, and cell functions at the single-cell level,

but it also allows the collection of a statistically relevant amount of data

from a heterogeneous cell population. In any efficient flow cytometry

workflow, the instrumentation and reagents must work together to

ensure reproducible, high-quality data. Here we describe the Invitrogen™

eBioscience™ Super Bright antibody conjugates, optimized for use in

flow cytometry and designed to provide exceptionally bright readouts

for better discrimination of cell subpopulations (Figures 1 and 2).

Super Bright polymer dyesThe Super Bright dyes are polymer-based fluorophores that are excited

by the violet laser (405 nm) and named according to their emission

wavelength (Figure 1). In addition to their very bright fluorescence

emission, the Super Bright antibody conjugates are fully compatible

with other fluoro phores commonly used in flow cytometry—including

R-phycoerythrin (PE), allophycocyanin (APC), and Invitrogen™ Alexa

Fluor™ and eFluor™ dyes—as well as with Invitrogen™ eBioscience™

buffers and fixatives and Invitrogen™ UltraComp eBeads™ micro-

spheres. These features, combined with our extensive portfolio of cell

Figure 2. 10-color T cell subset panel. Human peripheral blood cells were diluted with Invitrogen™ eBioscience™ Super Bright Staining Buffer (Cat. No. SB-4400-42) and

surface stained with the indicated reagents. Samples were then fixed and permeabilized according to the Invitrogen™ eBioscience™ Foxp3/Transcription Factor Staining

Buffer Set (Cat. No. 00-5523-00) protocol and stained with the indicated intracellular reagents. The fluorescent antibody panel included: anti-CD3 (clone OKT3) Super Bright

645, anti-CD4 (clone RPA-T4) eFluor 506, anti-CD8a (clone RPA-T8) APC-eFluor 780, anti-CD20 (clone 2H7) PE-Cyanine5.5, anti-CD25 (clone BC96) Super Bright 600,

anti-CD27 (clone O323) Super Bright 436, anti-CD183 (CXCR3) (clone CEW33D) PE-eFluor 610, anti-CD185 (CXCR5) (clone MU5UBEE) PE-Cyanine7, anti-Foxp3 (clone

PCH101) FITC, and anti-TIGIT (clone MBSA43) APC. Analysis was performed to discriminate various T cell subpopulations.

300 400 500 600 700 800 9000

20

40

60

80

100Super B

right 436

Super Bright 600

Super Bright 645

Super Bright 702

Fluo

resc

ence

Wavelength (nm)

CD

20 P

E-C

yani

ne5.

5

CD8 APC-eFluor 780CD3 Super Bright 645

Foxp

3 FI

TC

CD

25 S

uper

Bri

ght

60

0

CD4 eFluor 506 CD4 eFluor 506

CD

4 eF

luo

r 50

6

TIG

IT A

PC

CD27 Super Bright 436CD185 PE-Cyanine7 CD4 eFluor 506

Foxp

3 FI

TC TIGIT APC

No

rmal

ized

to m

od

e

CD3+

CD4+

CD8+

CD25+

CD25–

T cells

LN-homing T cells

CD4 T helper cells

Treg cells

Th1 in�ammatory CD4 T cells

CD

183

PE

-eF

luo

r 61

0

Mature/activated

Figure 1. Emission spectra of Super Bright 436, Super Bright 600, Super Bright

645, and Super Bright 702 polymer dyes. The black bar indicates the excitation

wavelength of the violet laser (405 nm). The less-intense, brown curve under the

blue emission shows the contribution of the (donor) Super Bright 436 dye to the

emission curves of the three tandem Super Bright polymer dyes.


BIOPrOBEs 75 TOOLs FOr FLOW CYTOMETrY

analysis reagents, provide many choices when designing multicolor

flow cytometry workflows, while also allowing you to expand the utility

of your violet laser.

The Super Bright 436 dye has an excitation maximum of 414 nm

and an emission peak of 436 nm (Figure 1). We recommend using a

450/50 nm bandpass filter or equivalent, similar to that used for detection

of eFluor 450 conjugates. Super Bright 436 antibody conjugates are

brighter than eFluor 450 conjugates and can serve as an alternative for

Brilliant Violet™ 421 conjugates, providing similar resolution of positive

and negative populations (Figure 3). Stability studies indicate that the

Super Bright 436 dye exhibits minimal loss of fluorescence when cells

are exposed to formaldehyde fixative for up to 3 days or when cells

are exposed overnight to ambient light.

The Super Bright 600 dye is a tandem dye comprising the Super

Bright 436 dye and an acceptor dye that emits fluorescence at 600 nm

Figure 4. Staining performance and post-fixation stability of Super Bright 600

dye. (A) Direct comparison of mouse splenocytes stained with CD8a antibody (clone

53-6.7) conjugated to either Super Bright 600 dye (red histogram) or Brilliant Violet

605 dye (gray histogram), at the same antibody concentration. (B) Mouse splenocytes

stained with CD45R antibody (clone RA3-6B2) conjugated to Super Bright 600 dye

and left unfixed (red histogram), or fixed in Invitrogen™ eBioscience™ IC Fixation Buffer

for 30 min (blue histogram), 24 hr (orange histogram), or 3 days (green histogram).

Cou

nts

A B

CD45R (clone RA3-6B2)CD8a (clone 53-6.7)

Cou

nts

Figure 3. Fluorescence intensity comparison with Super Bright 436 dye.

(A) Human peripheral blood cells stained with CD19 antibody (clone HIB19) con-

jugated to either Super Bright 436 dye (purple histogram), eFluor 450 dye (blue

histogram), or Brilliant Violet 421 dye (orange histogram) using the manufacturer’s

recommended volume per test (cells were gated on lymphocytes). (B) Human

peripheral blood cells stained with CD27 antibody (clone O323) conjugated to either

Super Bright 436 dye (purple histogram) or Brilliant Violet 421 dye (blue histogram).

Cou

nts

CD19 (clone HIB19)

A

CD27 (clone O323)

B

Cou

nts


(A) Mouse splenocytes stained with CD8a antibody (clone 53-6.7) conjugated to

Super Bright 645 dye (red histogram) or Brilliant Violet 650 dye (gray histogram), at

the same concentration of antibody. (B) Human peripheral blood cells stained with

CD8a antibody (clone RPA-T8) conjugated to Super Bright 645 dye (red histogram)

or Brilliant Violet 650 dye (gray histogram), using the same concentration of antibody.

Cou

nts

CD8a (clone 53-6.7)

A

CD8a (clone RPA-T8)

B

Cou

nts


(A) Mouse splenocytes stained with CD4 antibody (clone GK1.5) conjugated to

Super Bright 702 dye (red histogram) or Brilliant Violet 711 dye (gray histogram), at

the same concentration of antibody. (B) Human peripheral blood cells stained with

CD19 antibody (clone HIB19) conjugated to Super Bright 702 dye (red histogram) or

Brilliant Violet 711 dye (gray histogram), using the same concentration of antibody.

Cou

nts

Anti-Mouse CD4 (GK1.5)

A

Anti-Human CD19 (HIB19)

B

Cou

nts

(Figure 1). It can be detected using a 610/20 nm bandpass filter or

equivalent. Super Bright 600 conjugates are comparable in brightness to

Brilliant Violet™ 605 conjugates (Figure 4). The Super Bright 600 dye is

stable for up to 3 days when stored in a formaldehyde fixative solution.

Super Bright 645 is a tandem dye consisting of Super Bright 436

and an acceptor dye that has an emission peak of 645 nm (Figure 1).

It can be detected using a 660/20 nm bandpass filter or equivalent.

This tandem polymer dye is comparable in brightness to Brilliant Violet™

650 dye (Figure 5) and demonstrates less spillover into other violet

channels. Super Bright 645 is stable for up to 3 days when stored in

a formaldehyde fixative solution.

Super Bright 702 is a dye consisting of Super Bright 436 and an

acceptor dye that has an emission peak of 702 nm (Figure 1). It can

be detected using a 710/50 nm bandpass filter or equivalent, similar to

Brilliant Violet™ 711 dye. Super Bright 702 antibody conjugates



Table 1. Attune NxT Flow Cytometer configuration using 6 fluorescence detectors for the violet laser.

Laser

Fluorescence detectors available

2-laser configuration



Violet, 405 nm 6 6 6

Blue, 488 nm 2 2 2

Yellow, 561 nm NA NA 3

Red, 637 nm NA 3 3

Total fluorescence detectors available per configuration

8 11 14

Total parameters per configuration (includes FSC and SSC)

10 13 16

Table 2. Fluorophore guidelines for the 6 fluorescence detectors of the violet laser in the Attune NxT Flow Cytometer.

Detector Bandpass (nm) Fluorophores

VL1 450/40 Super Bright 436, Brilliant Violet™ 421, eFluor™ 450, Pacific Blue™, BD Horizon™ V450, VioBlue™

VL2 525/50 eFluor™ 506, Brilliant Violet™ 510, Pacific Green™, BD Horizon™ V500, VioGreen™

VL3 610/20 Super Bright 600, Brilliant Violet™ 605, Pacific Orange™

VL4 660/20 Super Bright 645, Brilliant Violet™ 650

VL5 710/50 Super Bright 702, Brilliant Violet™ 711

VL6 780/60 Brilliant Violet™ 786

Figure 7. Super Bright Staining Buffer minimizes nonspecific interactions.

Human peripheral blood cells were stained with CD8a antibody (clone RPA-T8)

conjugated to Super Bright 600 and CD4 antibody (clone SK3) conjugated to

Super Bright 436 (A, B) or Brilliant Violet 421 dye (C, D). Cells were stained in the

presence of Invitrogen™ eBioscience™ Flow Cytometry Staining Buffer only (A, C) or

Super Bright Staining Buffer was added to cells prior to addition of antibodies (B, D).

CA

CD4 Brilliant Violet 421

CD

8a S

uper

Brig

ht 6

00

CD

8a S

uper

Brig

ht 6

00

B

CD

8a S

uper

Brig

ht 6

00

CD4 Super Bright 436

CD

8a S

uper

Brig

ht 6

00

CD4 Brilliant Violet 421CD4 Super Bright 436

D

are similar in brightness to those of Brilliant Violet 711 dye (Figure 6)

and feature less spillover into the Brilliant Violet™ 786 channel. Fixation

compatibility is similar to the other Super Bright conjugates.

Learn more about these Super Bright polymer dyes and search

our ever-expanding portoflio of Super Bright antibody conjugates at

thermofisher.com/superbrightbp75.

Super Bright staining bufferSimilar to traditional fluorescent conjugates, Super Bright antibody

conjugates can be employed in most flow cytometry applications

without adjusting protocols. However, if two or more Super Bright

conjugates are combined in the same panel, we recommend using

Invitrogen™ eBioscience™ Super Bright Staining Buffer to minimize any

nonspecific interactions that may occur between these polymer-based

dyes (Figure 7). No special buffer is required when using a single

Super Bright conjugate within a panel that does not contain other

polymer dye conjugates. When Super Bright conjugates are used

in combination with other polymer dye conjugates, such as Brilliant

Violet dyes, the Super Bright Staining Buffer can also be used to help

reduce dye–dye interactions. Super Bright Staining Buffer is formulated

for use at 5 μL/test, making it convenient when preparing cocktails.

Expand the utility of the violet laserThe Invitrogen™ Attune™ NxT Flow Cytometer, introduced with up

to 4 lasers and 16 parameters of detection, will soon be available

with 6 fluorescence detectors for the violet laser, allowing measure-

ment of up to 8 fluorescent parameters with a 2-laser instrument,

11 fluorescent parameters with a 3-laser instrument, or 14 fluorescent

parameters with a 4-laser instrument (Table 1). The 6-channel violet

laser configuration, available on the 2-, 3-, and 4-laser instruments,

will accommodate the Super Bright polymer dye antibody conjugates

along with other commercially available violet-excitable fluorophores

(Table 2). See our comprehensive suite of products for flow cytometry,

from instruments and standards to antibodies and cell function reagents

at thermofisher.com/flowcytometrybp75. ■



Powering up drug discoveryCancer biology applications using the Attune NxT Flow Cytometer.

Flow cytometry is fast becoming a transformative tool in all phases of

the drug discovery process. The ability to examine individual cells at

the rate of thousands per second has made flow cytometry an attrac-

tive technology for investigating new drug candidates, evaluating the

effectiveness of cancer treatments, and understanding mechanisms

of cell health [1,2]. Continued advances in detection technologies

offer enhanced efficiencies in identifying and characterizing potential

cancer therapies. With its high sampling rates, fast detection speeds,

and clog resistance, the Invitrogen™ Attune™ NxT Flow Cytometer

enables accurate cell measurements for the evaluation of cancer drugs.

And because it allows the simultaneous detection of up to 14 colors

within a single sample, the Attune NxT Flow Cytometer is well suited

to multiparametric analyses.

Dose-response studies with the Attune NxT Flow Cytometer and AutosamplerFlow cytometry is an effective technique for dose-response studies,

providing quantitative data at the individual cell level on a large sample

size across a wide range of drug concentrations in a short amount

of time. Traditional cytometry has proven to be ineffective in many

high-throughput protocols because most flow cytometers rely solely

on hydrodynamic focusing technology, which cannot handle large,

clumpy cells without clogging the system. These clogs lead to cell

loss and machine downtime and require user intervention to resolve

issues. However, the fluidic system of the Attune NxT Flow Cytometer

was designed to minimize the potential for clogging and sample loss

and, because it is syringe pump driven, has an automated function

to unclog the flow cell if needed. Moreover, the acoustic-assisted

hydrodynamic focusing technology employed by the Attune NxT Flow

Cytometer effectively interrogates cells at high sampling rates, even

in dilute samples, enabling the use of reduced sample preparation

procedures.

The application note “Flow cytometry analysis of dose-response

for apoptosis induction” provides an easy and efficient protocol

for evaluating therapeutic cancer drugs using the Attune NxT Flow

Cytometer together in conjunction with the Attune Autosampler, which

allows for rapid processing of multiple samples in 96-well plates. In

this protocol, Jurkat cells (human T cell leukemia) are grown and

treated in 96-well plates with eight concentrations of four different

anti-cancer drugs—staurosporine, cycloheximide, camptothecin, and

etoposide (Figures 1 and 2)—and then evaluated for apoptosis using

the Invitrogen™ CellEvent™ Caspase-3/7 Green Detection Reagent and

analyzed on the Attune NxT Flow Cytometer (Figure 1). A total of 20,000

cells are acquired from each well at a speed of 200 µL/min.

log [Drug] (M)–7–5–4 –8–6

0

50

100Staurosporine

Cycloheximide

Camptothecin

Etoposide

Cel

lEve

nt C

asp

ase-

3/7

Gre

en s

tain

ed c

ells

(%)

Figure 1. Dose-response curves for evaluating the effectiveness of four

anti-cancer drugs in inducing apoptosis in Jurkat cells. Apoptosis was induced

in Jurkat cells (human T cell leukemia) using eight different concentrations of the

indicated drugs, and then detected using the Invitrogen™ CellEvent™ Caspase-3/7

Green Flow Cytometry Assay Kit (Cat. No. C10740). Each drug concentration

(shown from high to low on the x-axis) was run in triplicate. Statistics from the

whole plate were exported as a single CSV file and analyzed using GraphPad

Prism™ statistical software.

Figure 2. Results displayed with the Attune NxT software heat map. The

Invitrogen™ Attune™ NxT Flow Cytometer software produces a heat map view that

provides a quick visual evaluation of the plate results. This view uses a range of

colors along with a numerical value to indicate the percentage of cells in each well

that were labeled with a particular probe, (e.g., apoptotic cells, as indicated by

a positive signal from the Invitrogen™ CellEvent™ Caspase-3/7 Green Detection

Reagent). The percent of positive-stained cells is displayed in each well, and the

well color transitions from purple to red as the percentage increases, with red wells

containing the highest percentage of apoptotic cells.



Additional time savings are obtained by adding

the CellEvent Caspase-3/7 Green Detection

Reagent directly to live cells in media, elimi-

nating the need to wash, fix, or permeabilize

the cells prior to analysis. Also, the Attune

NxT software produces a heat map view that

provides a quick visual evaluation of the plate

results, allowing you to simultaneously visualize

multiple variables in the experiment (Figure 2).

Cell health assessment with the Attune NxT Flow CytometerMulticolor flow cytometry provides a means

of analyzing large numbers of individual cells

for multiple cell health parameters in a short

amount of time. For example, the Attune

NxT Flow Cytometer enables the simultane-

ous measurement of up to 16 parameters

(14 different emission channels plus for-

ward and side scatter) of cell health at the

single-cell level, with sampling rates of up

to 35,000 cells/sec at 1 mL/min. This high

sampling rate allows for thousands to millions

of cells to be analyzed in minutes.

The application note entitled “Life, death,

and cell proliferation: Multiplex single-cell

analysis with minimal compensation” combines

proliferation, viability, and apoptosis functional

assays (Table 1) in a single experimental

design to simultaneously examine a CD4+ sub-

set of stimulated human T lymphocytes. This

protocol requires minimal setup. In addition,

fluorescence color compensation requirements

can be minimized or even eliminated by using

the Attune NxT Flow Cytometer equipped with

405 nm violet, 488 nm blue, 561 nm yellow,

and 637 nm red lasers, in conjunction with

spectrally compatible fluorophores that show

minimal spectral overlap.

The Invitrogen™ LIVE/DEAD™ Fixable

Violet Dead Cell Stain (405 nm violet laser)

Figure 3. Data acquisition on the Attune NxT Flow Cytometer for stimulated human lymphocytes. (A) Gating

on density plot of SSC-height (SSC-H) vs. SSC-width (SSC-W) removes cell aggregates from the analysis. The

singlet population is displayed in (B) through (E). (B) Histogram of Invitrogen™ LIVE/DEAD™ Fixable Violet Dead

Cell Stain (Cat. No. L34963) fluorescence. The large peak on the left is the population of live cells; whereas the

smaller peak on the right represents the dead cell population. (C) Histogram of Invitrogen™ CellEvent™ Caspase-3/7

Green Detection Reagent (Cat. No. C10740) fluorescence. The large peak on the right is the apoptotic cell popu-

lation. (D) Histogram of R-phycoerythrin (PE) anti-CD4 antibody (Cat. No. MHCD0404-4) fluorescence showing

CD4+ and CD4– cells. (E) Histogram of Invitrogen™ CellTrace™ Far Red Cell Proliferation Dye (Cat. No. C34572)

fluorescence, showing the individual generations of proliferating T cells. The analysis of the cell health of these

stimulated human lymphocytes continues in Figure 4.

provides a simple method of distinguishing live and dead cells using an amine-reactive dye

that maintains identification of dead cells after fixation. The Invitrogen™ CellTrace™ Far Red

Cell Proliferation Dye (637 nm red laser) facilitates the identification of each generation of cells

based on fluorescence intensity. The combination of the LIVE/DEAD Fixable Violet viability dye,

the CellTrace Far Red dye, and the CellEvent Caspase-3/7 Green apoptosis indicator (488 nm

Table 1. Three spectrally compatible cell viability assays for characterizing cell populations by flow cytometry.

Kit DyeLaser, bandpass filter Function Utility

CellTrace™ Far Red Cell Proliferation Kit

Cell-permeant amine-reactive dye

637 nm red laser, 670/14 nm BP

Covalently binds to amine groups, allowing the determination of different cell generations based on fluorescence intensity

When all three probes are used together, three separate cell populations can be identified—live cells, proliferating cells, and apoptotic cells. For more information, go to thermofisher.com/ flow-cellviability

LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit

Cell-impermeant amine-reactive dye

405 nm violet laser, 440/50 nm BP

Allows identification of dead cells before and after fixation based on membrane integrity

CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit

Cell-permeant fluorogenic caspase substrate

488 nm blue laser, 530/30 nm BP

Enables detection of activated caspase-3 and -7 in apoptotic cells

Singlets

BA C

SS

C-H

(x 1

0 3)

100 200 300 4001

SSC-W

750

0

500

103102

LIVE/DEAD Fixable Violet �uorescence

4

2

0105

250 Cou

nts

(x 1

0 3)

103 104102

CellEvent Caspase 3/7 �uorescence

20

10

0

Cou

nts

(x 1

0 3)

CD4 RPE �uorescenceC

ount

s (x

10

3)

CellTrace Far Red �uorescence

3

Cou

nts

(x 1

0 3)

102 103 104 105 1060

2

–103 0 103 104 105

0

4

8

6

2

D E

105101

1

104

All cells Singlet cells Singlet cells

Singlet cells Singlet cells



References1. Sharma P, Muthuirulan P (20 September 2016).

Flow cytometry: breaking bottlenecks in drug discovery and development. Drug Target Review. Retrieved from drugtargetreview.com/article/14512/flow-cytometry-breaking-bottle-necks-drug-discovery-development/

2. Thayer M, Draper D, Saims D et al. (2016) In depth myeloid cell characterization in the murine syngeneic CT26 colon carcinoma model by 10 color flow cytometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Associ-ation for Cancer Research; 2016 Apr 16–20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 76(14 Suppl): Abstract 3242. doi: 10.1158/1538-7445.AM2016-3242.


Attune™ NxT Flow Cytometer—Blue/Red/Violet/Yellow Lasers 1 each A24858

Attune™ Autosampler 1 each 4473928

CellTrace™ Far Red Cell Proliferation Kit 20 reactions180 reactions

C34572C34564

LIVE/DEAD™ Fixable Violet Dead Cell Stain 80 assays200 assays400 assays

L34963L34955L34964

CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit 20 assays100 assays

C10740C10427

Anti–Human CD4 Mouse Monoclonal Antibody (clone S3.5), PE conjugate 2 mL MHCD0404-4

AbC™ Total Antibody Compensation Bead Kit 25 tests100 tests

A10513A10497

ArC™ Amine-Reactive Compensation Bead Kit 25 tests100 tests

A10628A10346

Figure 4. Data acquisition on the Attune NxT Flow Cytometer for stimulated human lymphocytes. Cells

were first gated on the singlet cell population as shown in Figure 3. (A) Density plot of singlet population showing

the discrimination of live and dead cells using Invitrogen™ LIVE/DEAD™ Fixable Violet Dead Cell Stain (Cat. No.

L34963) fluorescence. A gate was created around the live cells, which represent 67.7% of the singlet population.

(B) Density plot of Invitrogen™ CellEvent™ Caspase-3/7 Green Detection Reagent (Cat. No. C10740) fluorescence

vs. Invitrogen™ CellTrace™ Far Red Cell Proliferation Dye (Cat. No. C34572) fluorescence. Fewer apoptotic cells

are seen in early generations (upper right), whereas far more apoptotic cells are seen in later generations (upper

left). (C) Density plot of CellEvent Caspase-3/7 Green fluorescence vs. LIVE/DEAD Fixable Violet fluorescence.

Live cells (26.5%), dead cells (34.6%), and apoptotic cells (38.3%) can be identified. The live cells identified in (A)

were further analyzed: (D) Gated on the live cells from (A), a histogram of live singlet cells labeled with CellTrace

Far Red dye, showing multiple peaks representing each generation of this proliferating population. (E) Gated on

the live cells from (A), a density plot of PE anti-CD4 antibody fluorescence (labeling T helper lymphocytes) vs.

CellTrace Far Red fluorescence, showing the increasing generational proliferation of CD4+ cells.

Live cells67.7%

Dead34.6%

Apoptotic38.3%

Live26.5%

A B C

ED

103 104102

Cel

lEve

nt C

asp

ase-

3/7

Gre

en �

uore

scen

ce

105

CellTrace Far Red �uorescence102 103 104 105 106102 103 104 105 106

0

2

103 104102


700

250

0105

SS

C-H

(x 1

0 3)

500


105

104

103

102

101

103 104102 105

105

104

103

102

101

CD

4 P

E �

uore

scen

ce

104

103

102

101



Cou

nts

(x 1

0 3)

Cel

lEve

nt C

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ase-

3/7

Gre

en �

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scen

ce

1

Singlet cells Singlet cells Singlet cells

Live singlet cells Live singlet cells

blue laser) enables the identification of three separate cell populations—live, proliferating, and

apoptotic cells; the PE anti-CD4 antibody conjugate (561 nm yellow laser) was added to these

functional probes to identify the CD4+ population during the analysis.

With this experimental design, apoptotic cells can easily be discriminated from live and necrotic

cells (Figure 3). Compared with unstimulated cells, stimulated cells have a higher percentage of

apoptotic cells, demonstrating the activation

of the caspase enzymes and the early stages

of apoptosis. Using the PE anti-CD4 antibody

conjugate, we found that the majority of the

proliferating cells, as identified by the CellTrace

Far Red reagent, are CD4+ (Figure 4).

Streamline workflows with the Attune NxT Flow CytometerThe Attune NxT Flow Cytometer was designed

to facilitate high-throughput screening assays

in drug discovery applications. The Attune NxT

acoustic focusing technology simplifies sam-

ple preparation requirements, provides rapid

throughput rates that are up to 10 times faster

than traditional flow cytometers, and offers the

Attune Autosampler option to accommodate

96-well (and 384-well) plates. The increased

data fidelity, faster acquisition rates, and

improved fluidics expand the scope and

power of flow cytometry into areas such

as cancer, synthetic biology, and drug dis-

covery. Together with our wide selection of

flow cytometry antibodies and reagents, the

Attune NxT Flow Cytometer and Autosampler

streamline high-throughput workflows, sav-

ing time and expanding resources. Visit

thermofisher.com/flowappnotesbp75 to

download these application notes. ■



Evaluate both RNA and protein targets in single cellsPrimeFlow RNA assay for detecting RNA targets by flow cytometry.

Flow cytometry has long been the standard

for characterizing heterogeneous cell popu-

lations, thanks to its ability to rapidly acquire

and analyze millions of individual cells and

to multiplex and detect both cell-surface

and intracellular proteins in a straightforward

workflow. One major limitation, however, is

the availability of antibodies to measure all

analytes of interest. Researchers studying

microRNA (miRNA), long noncoding RNA

(lncRNA), messenger RNA (mRNA), viral tran-

scripts (vRNA), unique model organisms, or

other targets for which antibody development

is troublesome have not been able to utilize

the power of flow cytometry. Historically they

have had to conduct numerous independent

experiments, often on bulk samples, to ana-

lyze these targets.

With the Invitrogen™ PrimeFlow™ RNA

Assay Kit, researchers can now reveal the

dynamics of RNA transcription together with

protein expression patterns by flow cytom-

etry. The PrimeFlow RNA assay employs

fluorescence in situ hybridization (FISH) with

branched DNA (bDNA) signal amplification for

the simultaneous detection of up to four RNA

targets, and it can be used in combination with

immunolabeling of both cell-surface and intra-

cellular proteins using fluorophore-conjugated

antibodies.

Detect and amplify RNA targets with the PrimeFlow RNA assayIn the PrimeFlow RNA assay workflow, cells

are first labeled with cell-surface antibodies,

fixed and permeabilized, and then labeled with

intracellular antibodies. These cells are then

sequentially hybridized with probes specific

for the RNA targets, and hybridized targets are detected after bDNA signal amplification. In the

initial hybridization step, a gene-specific oligonucleotide target probe set that contains 20–40

probe pairs (or a single pair in the case of miRNA probe sets) binds to the target RNA sequence.

An individual probe pair contains two oligonucleotides that are designed to bind adjacent to

each other on the RNA transcript for bDNA signal amplification to take place.

bDNA signal amplification is achieved through a series of sequential hybridization steps with

preamplifiers, amplifiers, and then fluorophore-conjugated label probes. The preamplifier mole-

cules confer an additional level of specificity because they will hybridize to the RNA target only

after both members of the oligonucleotide target probe set have bound to their target sequence.

Multiple amplifier molecules subsequently hybridize to their respective preamplifier molecules.

Finally, label probe oligonucleotides, which are conjugated to a fluorescent dye, hybridize to

their corresponding amplifier molecules. A fully assembled signal amplification “tree” has 400

label probe binding sites. When all target-specific oligonucleotides in the probe set bind to the

target RNA transcript, 8,000- to 16,000-fold amplification can be achieved.

Measure up to 4 RNA targets per cell by flow cytometryThe PrimeFlow RNA assay currently offers four unique amplification structures that allow simul-

taneous measurement of up to four different RNA targets for multicolor flow cytometry analysis.

Alexa Fluor™ 647 (Type 1) and Alexa Fluor™ 568 (Type 10) probe sets provide the most sensitive

detection of the four sets and should be used for target genes with low or unknown levels of

Figure 1. The PrimeFlow RNA assay workflow. The workflow for the Invitrogen™ PrimeFlow™ RNA Assay Kit

(Cat. No. 88-18005-204) starts with optional antibody labeling, followed by fixation and permeabilization, and

then hybridization with gene-specific target probes. This hybridization is then detected after branched DNA

(bDNA) signal amplification using preamplifiers, amplifiers, and label probes. Labeled cells are analyzed on a

standard flow cytometer.

Ampli�er

Gene 1

Gene 2

Process cells using a �ow cytometer

Label probes

Add �uorescently labeled probes to cells

Preampli�er

Suspension cells with �xed RNA

Label proteins with antibody (optional)

Fix and permeabilize cellsin suspension

Label intracellular proteins with antibodies (optional)

Incubate cells with gene-speci�c probe sets

Hybridize with preampli�erand ampli�er DNA

Gene-speci�clabel extenders (LE)

Sample preparation Target hybridization Signal ampli�cation Detection

CD8 PE-Cyanine7

CD

8 m

RN

A A

lexa

Flu

or

647



expression. Alexa Fluor™ 488 (Type 4) and Alexa Fluor™ 750 (Type 6)

probe sets are designed for detecting target genes with medium to

high levels of expression. The lower sensitivity of Alexa Fluor 488 probe

sets, as compared with Alexa Fluor 647 probe sets, is in part due to

potentially high levels of cell autofluorescence at the Alexa Fluor 488

detection wavelengths.

Once RNA targets have been hybridized and amplified using the

PrimeFlow RNA Assay Kit, the cells can be analyzed on a standard

flow cytometer. Figure 1 illustrates the workflow for the simultaneous

detection of two unique RNA targets. With target-specific probe sets,

the PrimeFlow RNA assay can be used to detect miRNA, lncRNA, and

mRNA, as well as vRNA and telomere DNA.

miRNA detection at the level of single cellsApproximately 75% of the human genome can be transcribed into RNA;

however, only 1.5% of the human genome codes for mRNA, which

is typically translated into protein. Even though they do not encode

proteins, nontranslated (or noncoding) RNA sequences often show

tissue-specific expression and may contain sequences conserved

across species, suggesting that they play a role in cell function. For

example, miRNA sequences serve as key translational regulators for

30% of all protein-coding genes in diverse biological processes [1,2].

With advances in transcriptomic techniques, researchers have

been able to identify, profile, validate, and functionally analyze relevant

noncoding RNA in different models and diseases. However, analysis

at the single-cell level, especially for miRNA, has been limited by low

sensitivity and poor resolution. For detection of miRNA and other small

Figure 2. PrimeFlow RNA assay detection of miR-146a, Arg1 mRNA, Cxcl13 mRNA, and Retnla mRNA in mouse peritoneal cells. C57BI/6 mouse resident perito-

neal exudate cells were analyzed using the Invitrogen™ PrimeFlow™ RNA Assay Kit (Cat. No. 88-18005-204). Cells were stained with eFluor™ 450 anti–mouse F4/80 and

PE-Cyanine7 anti–mouse CD11b antibodies, and fixed and permeablized using PrimeFlow RNA Assay Kit buffers and protocols. Next, cells were hybridized with target-specific

oligonucleotides using Type 1 human/mouse miR146a Alexa Fluor™ 647, Type 4 mouse Arg1 (arginase 1) Alexa Fluor™ 488, Type 6 mouse Cxcl13 Alexa Fluor™ 750, and

Type 10 mouse Retn1a (Relm α) Alexa Fluor™ 568 target probes. Viable CD11b+ cells were used for analysis. The data show that both small peritoneal macrophages (SPM,

F4/80–) and large peritoneal macrophages (LPM, F4/80+) were positive for miR-146a. SPM expressed high levels of Retnla (Relm α) mRNA, whereas LPM were positive for

Cxcl13 mRNA and expressed low levels of Arg1 (arginase 1) mRNA.

RNA, we recommend the use of the Invitrogen™ PrimeFlow™ microRNA

Pretreatment Buffer and the accompanying protocol. The use of this

buffer with the PrimeFlow RNA assay improves the retention of some

small RNA targets, resulting in better detection sensitivity. Figure 2

demonstrates that the PrimeFlow RNA Assay Kit, in combination with

the PrimeFlow microRNA Pretreatment Buffer, enables the detection of

miRNA together with antibody-based immunophenotyping.

Learn more about the PrimeFlow RNA assayThe ability to measure RNA and protein expression together in individ-

ual cells provides a means of correlating their levels with cell function

over time or in response to a stimulus. The PrimeFlow RNA Assay Kit

provides a complete buffer system, compensation kit, and reagents

for detecting up to 4 RNA transcripts in mammalian cells optionally

labeled with antibodies that recognize cell-surface or intracellular pro-

teins. For more information, including key publications and customer

webinars as well as catalog probe sets and ordering guidelines, visit

thermofisher.com/primeflowbp75. ■


PrimeFlow™ RNA Assay Kit 40 tests100 tests

88-18005-20488-18005-210

PrimeFlow™ microRNA Pretreatment Buffer 100 tests 88-18006

F4/80 eFluor 450

miR

-146

a A

lexa

Flu

or 6

47

F4/80 eFluor 450 F4/80 eFluor 450 F4/80 eFluor 450

Arg

1 m

RN

A A

lexa

Flu

or 4

88

Cxc

l13

mR

NA

Ale

xa F

luor

750

Ret

nla

mR

NA

Ale

xa F

luor

568

References1. Lewis BP, Burge CB, Bartel DP (2005) Cell 120:15–20.

2. Felekkis K, Touvana E, Stefanou Ch et al. (2010) Hippokratia 14:236–240.


JOUrNAL CLUB BIOPrOBEs 75

Coming ’round to spheroid cultureMandavilli BS, Neeley C, Bates M, Persmark M (2015) BioProcessing Journal 14:44–49.

From developmental studies to cancer biology,

expanding applications for 3D cell culture have

triggered the development of new reagents

and techniques to support these diverse

research fields. Spheroids (or sphere cultures)

are an important subset of 3D in vitro cultures

that can mimic several characteristics of in

vivo normal and malignant tissues, including

cell–cell interactions, nutrient gradients, and

diffusion kinetics, as well as gene expression

profiles [1,2]. Mandavilli and co-workers have

published an article in BioProcessing Journal

that provides an overview of the history, chal-

lenges, and potential of spheroid culture. Here

is a brief summary of their article.

The extracellular matrixThe extracellular matrix (ECM), typically

composed of soluble proteins and insoluble

collagen fibers, not only provides the scaf-

folding for complex tissues but also plays a

role in signaling, differentiation, and homeo-

stasis. When culturing spheroids, the ECM

adhesion proteins can interfere with spheroid

formation by interacting with the culture vessel

surface, leading to the development of satellite

colonies. Figure 1 shows a time course of

spheroid development in untreated plates vs.

Thermo Scientific™ Nunclon™ Sphera™ plates,

which are coated with a recently developed

hydrophilic polymer that promotes the growth

of spheroids without satellite colonies.

In addition to the development of culture

vessels, several types of engineered ECMs are

now available for use in 3D culture systems.

See “Cancer biology in the third dimension”

on page 11 for a discussion of 3D cultures

grown in a tissue-like matrix.

The role of oxygen in cell cultureAs with 2D cell culture, culturing spheroids requires precisely controlled abiotic conditions,

including temperature, pH, and oxygen and carbon dioxide levels, in addition to specialized

equipment such as coated culture vessels. Over two decades ago, researchers found that

both embryonic tissue culture and fibroblast tissue cultures were more successful when grown

under controlled oxygen conditions that more closely resembled those found in the human body

(1%–12%), rather than those found in the atmosphere (20%). Cells cultured under these hypoxic

Figure 1. Comparison of cancer spheroid formation using Nunclon Sphera plates vs. untreated plates

containing methylcellulose. HCT 116 cells, maintained in Thermo Scientific™ Nunclon™ Delta cell culture flasks

(Cat. No. 136196), were seeded in Thermo Scientific™ Nunclon™ Sphera™ 96-well U-bottom plates (Cat. No.

174925) at densities of 100–3,000 cells/well in 200 μL/well Gibco™ DMEM (with high glucose, GlutaMAX™

supplement, and pyruvate; Cat. No. 10569-010) containing 10% FBS (Cat. No. A3382001), 1X MEM Non-

Essential Amino Acids (Cat. No. 11140-050), 100 U/mL penicillin–streptomycin, and 25 mM HEPES. Traditional

plates not treated for cell or tissue culture (untreated plates) were similarly seeded in complete DMEM medium

that also contained 3% methylcellulose. Plates were briefly centrifuged at 250 x g for 5 min and then incubated

at 37°C and 5% CO2; cells were re-fed every 72 hr by carefully removing 100 μL of medium from each well and

replenishing with 100 μL of fresh medium. Formation and growth of spheroids were imaged. (A) After 112 hr

incubation, cancer spheroids grown in Nunclon Sphera plates show more uniform shape, better-defined edges,

and cleaner backgrounds than those grown in untreated plates at all seeding densities. (B) At a seeding density

of 100 cells/well, the early time-course of spheroid formation in the Nunclon Sphera plate reveals spheroids after

only 18 hr and many fewer satellite colonies than in the untreated plate. Images used with permission from Helmut

Dolznig, Institute of Medical Genetics, Medical University of Vienna.

100 500 1,000 3,000

0 hr 18 hr 42 hr 112 hr

Number of cells seeded per wellA

B

100

cells

/wel

l see

ded

Unt

reat

ed96

U-w

ell p

late

Unt

reat

ed96

U-w

ell p

late

Nun

clon

Sp

hera

96 U

-wel

l pla

teN

uncl

on S

phe

ra96

U-w

ell p

late


BIOPrOBEs 75 JOUrNAL CLUB

References1. Fennema E, Rivron N, Rouwkema J, et al. (2013) Trends Biotechnol 31:108–115.

2. Hirschhaeuser F, Menne H, Dittfeld C et al. (2010) J Biotechnol 148:3–15.

3. Parrinello S, Samper E, Krtolica A et al. (2003) Nat Cell Biol 5:741–747.

4. Atkuri KR, Herzenberg LA, Niemi AK et al. (2007) Proc Natl Acad Sci USA 104:4547–4552.

5. Lengner CJ, Gimelbrant AA, Erwin JA et al. (2010) Cell 141:872–883.


EVOS™ FL Auto Imaging System 1 each AMAFD1000

EVOS™ Onstage Incubator 1 each AMC1000

FluoroBrite™ DMEM 500 mL10 x 500 mL

A1896701A1896702

Image-iT™ Hypoxia Reagent 1 mg H10498

NucBlue™ Live ReadyProbes™ Reagent 6 x 2.5 mL R37605

Nunclon™ Sphera™ Microplate, round-bottom 96-well plates

8 each 174925

Figure 2. Detection of hypoxia in cells growing in 3D culture. A549 cells were incubated in Gibco™ FluoroBrite™ DMEM (Cat. No. A1896701) with 5 μM Invitrogen™

Image-iT™ Hypoxia Reagent (red, Cat. No. H10498) at different levels of oxygen—(A) 20%, (B) 5%, (C) 2.5%, and (D) 1%—for 1 hr on the Invitrogen™ EVOS™ Onstage

Incubator and then imaged using the Invitrogen™ EVOS™ FL Auto Imaging System. The red-fluorescent hypoxia signal can be detected when oxygen levels drop below 5%.

A B C D

20% oxygen 5% oxygen 2.5% oxygen 1% oxygen

Figure 3. Assessment of the hypoxic core in a single HeLa spheroid. HeLa cells

(250 cells/well) were cultured on Thermo Scientific™ Nunclon™ Sphera™ 96-well

U-bottom plates (Cat. No. 174925) for 2 days in complete medium. The spheroids

were then incubated in situ with 5 μM Invitrogen™ Image-iT™ Hypoxia Reagent

(red, Cat. No. H10498) for 3 hr, and nuclei were counterstained with Invitrogen™

NucBlue™ Live ReadyProbes™ Reagent (blue, Cat. No. R37605). The stained

spheroids were transferred by pipetting using wide-bore pipette tips to a Thermo

Scientific™ Nunc™ Glass Bottom Dish (12 mm, Cat. No. 150680), and images were

taken on a confocal microscope.

conditions grew faster, lived longer, and showed lower levels of stress

[3–5]. A cell culture incubator that controls nitrogen, carbon dioxide,

and oxygen gas is the best way to achieve hypoxic conditions. The

Invitrogen™ EVOS™ FL Auto Imaging System with Onstage Incubator

includes an environmental chamber that allows for the precise control

of oxygen levels, temperature, and humidity over an extended period

of time and during live-cell imaging.

The capability to control oxygen levels during incubation and

imaging combined with the development of a real-time fluorogenic

indicator of intracellular oxygen levels has advanced both 2D and 3D

cell culture methods. The Invitrogen™ Image-iT™ Hypoxia Reagent is

a cell-permeant probe that begins to fluoresce when oxygen levels

fall below 5%, enabling sensitive and reproducible measurements of

hypoxia in cells (Figure 2). Unlike cells grown in 2D culture, spheroids

have a heterogeneous cell composition, with cells located at the surface

functioning differently from those buried in the middle. In particular,

spheroids possess the same hypoxic core seen in tumors, where cells

rapidly outgrow their blood supply, leaving the center of the tumor with

an extremely low oxygen concentration. These chronically hypoxic

regions are highly resistant to therapy as they are especially difficult to

penetrate with chemotherapy. The Image-iT Hypoxia Reagent has been

used to characterize the hypoxic core found in spheroids (Figure 3).

Spheroids for cancer biology researchIn addition to a hypoxic core, spheroids have been reported to

replicate other key elements of tumors, including necrosis, angiogen-

esis, and cell adhesion. Thus, spheroid culture has major implications,

not only for studying how the interplay between cells, tissues, and the

ECM affects pathological states, but also for the development of more

robust drug screening programs and improved organotypic models. ■


JOUrNAL CLUB BIOPrOBEs 75

Measurement and characterization of apoptosis by flow cytometryTelford W, Tamul K, Bradford J (2016) Curr Protoc Cytom 77:9.49.1–9.49.28.

Apoptosis plays a crucial role in the regulation of cell and tissue

homeostasis. Well characterized in the immune system, apoptosis

mechanisms are now informing anti-cancer therapies. Telford and

coauthors recently published a methods chapter providing detailed

protocols for several methods to measure and characterize apoptosis

using multiparametric flow cytometry. The article provides basic theory

discussions, detailed reagent listings, step-by-step procedures, and

guidance on analysis methods, while also identifying critical items to

consider when performing these assays.

Caspase assays highlightedA distinctive feature of the early stages of apoptosis is the activation of

caspase enzymes, which involves the cleavage of protein substrates.

Four assays for measuring caspase activation during cell death are

featured: three fluorescence-based assays each label unfixed cells with

a fluorogenic substrate (FAM-FLICA™, PhiPhiLux™ G1D2, or Invitrogen™

CellEvent™ Caspase-3/7 Green Detection Reagent (Figure 1)), and

the fourth assay uses an anti-caspase monoclonal antibody, which

requires that cells be fixed and permeabilized. Protocols for combining

each of the four caspase assays with fluorescent viability stains (e.g.,

Invitrogen™ SYTOX™ Dead Cell Stains and Invitrogen™ LIVE/DEAD™

Fixable Dead Cell Stains) and with fluorescent annexin V conjugates in a

multiparametric format are provided to more fully characterize cell death.

Critical considerationsNo one assay fully characterizes apoptosis, and these assays are

particularly powerful when combined to determine several charac-

teristics of apoptosis simultaneously. The multiparametric nature of

flow cytometry makes the technology ideally suited for measuring

apoptosis. As with any flow cytometry experiment, proper controls

are essential. A negative (or untreated) control, and a good positive

control, are required for proper data interpretation.

Understanding the kinetics of apoptosis is important, as the apop-

totic process is highly variable across cell types, and even within the

same cell type when cells experience different levels of activation. It is

important to measure the cell death process through time, as some cells

will die quickly whereas others follow a slower time course of cell death.

Care also should be taken when using scatter gating because exclusion

of dead and dying cells is not always obvious and submicron-level debris

may include information about apoptosis. Keep in mind that apoptosis

can be rapid and may continue during assay preparation; therefore, try

to keep the assay duration as short as possible. Processing steps like

vortex mixing, washing, and centrifugation may induce or accelerate

the apoptotic process and may damage or destroy apoptotic cells;

therefore, it is recommended that cells be treated gently during sample

preparation to avoid these unwanted effects.

Finally, the selected probes must be spectrally compatible and well

matched to the cytometer configuration. By choosing fluorescent probes

with minimal spectral overlap, color compensation can be minimized.

These relatively simple multiparametric assays are powerful techniques

for the evaluation and characterization of cell death. ■

Figure 1. Detecting active caspases and necrotic cells using the CellEvent

Caspase 3/7 Green Flow Cytometry Assay Kit. Jurkat cells (human T cell leukemia)

were treated with (A) DMSO or (B) 10 μM camptothecin for 3 hr before labeling

with the Invitrogen™ CellEvent™ Caspase 3/7 Green Flow Cytometry Kit (Cat. No.

C10427). Stained samples were analyzed on the Invitrogen™ Attune™ Flow Cytometer

equipped with a 488 nm laser, and fluorescence emission was collected using a

530/30 nm bandpass filter for CellEvent Caspase 3/7 Green Detection Reagent

and a 690/50 nm bandpass filter for Invitrogen™ SYTOX™ AADvanced™ Dead Cell

Stain (both stains are provided in the kit). The treated cells have a higher percentage

of apoptotic cells (B) than the basal level of apoptosis seen in the control cells (A).

A = apoptotic cells; V = viable cells; N = necrotic (or late apoptotic) cells.


CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit 100 assays C10427

LIVE/DEAD™ Fixable Dead Cell Stain Sample Kit 320 tests L34960

Pacific Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit

50 assays A35136

PO-PRO™-1 Iodide (435/455), 1 mM solution in DMSO 1 mL P3581

SYTOX™ Dead Cell Stain Sampler Kit, for flow cytometry 1 kit S34862

Vybrant™ FAM Caspase-3 and -7 Assay Kit, for flow cytometry

25 assays V35118

YO-PRO™-3 Iodide (612/631), 1 mM solution in DMSO 1 mL Y3607

V A

N

V

N

BA

–103 –102 103 104 105

–104

102

104

105

106

CellEvent Caspase-3/7 Green �uorescence

SY

TOX

AA

Dva

nced

�uo

resc

ence

–103

–103 –102 103 104 105

–104

102

104

105

106

CellEvent Caspase-3/7 Green �uorescence

SY

TOX

AA

Dva

nced

�uo

resc

ence

–103

Fluorophore and reagent selection guide for flow cytometry

Excitation laser

Common emission filters (nm)

Attune NxT channel (nm) Recommended dyes

Viability dyes (compatible

with fixation)Viability dyes (unfixed cells)

DNA content/ cell cycle dyes

(live cells)

DNA content/ cell cycle dyes

(fixed cells) Apoptosis dyes Cell proliferation dyes

Reactive oxygen species

(ROS) dyes Phagocytosis dyesFluorescent

proteins Other dyes

UV (~

350

nm)

369/28379/28 NA BD Horizon BUV395

440/40450/50 NA

Alexa Fluor 350

Alexa Fluor 405LIVE/DEAD

Fixable BlueDAPI

SYTOX Blue

Hoechst 33342

Vybrant DyeCycle Violet

FxCycle Violet (DAPI)Annexin V, Alexa Fluor 350

Annexin V, Pacific BlueClick-iT Plus EdU

Alexa Fluor 350ECFP BD Horizon BUV496

740/35 NA BD Horizon BUV737

Viol

et (4

05 n

m)

425/20440/50450/50455/50450/65460/50

VL1

440/50

Super Bright 436

eFluor 450

Pacific Blue

Alexa Fluor 405

LIVE/DEAD Fixable Violet

DAPI

SYTOX BlueVybrant DyeCycle

VioletFxCycle Violet

Annexin V, Pacific Blue

Annexin V, eFluor 450

PO-PRO-1

CellTrace Violet

Click-iT Plus EdU Pacific Blue

Azurite

Cerulean

TagBFP

ECFP

mTurquoise

AmCyan

BD Horizon BV421

BD Horizon V450

VioBlue

510/50512/25520/35525/50530/40542/50550/40

VL2

512/25

Pacific Green

eFluor 506LIVE/DEAD

Fixable AquaViolet Ratiometric Probe

(F2N12S)T-Sapphire

BD Horizon BV510

BD Horizon V500

VioGreen

585/40595/50605/40610/20

VL3

603/48

Super Bright 600

Pacific Orange

Qdot 605

LIVE/DEAD Fixable Yellow

Violet Ratiometric Probe (F2N12S)

BD Horizon BV570

BD Horizon BV605

BD Horizon BV650

660/20660/40710/50

VL4

710/50

Super Bright 645

Super Bright 702

Qdot 705

BD Horizon BV650

BD Horizon BV711

Blue

(488

nm

)

525/50525/30525/40530/30530/40

BL1

530/30

Alexa Fluor 488

FITCLIVE/DEAD

Fixable GreenSYTOX Green

Vybrant DyeCycle Green

Annexin V, Alexa Fluor 488

Annexin V, FITC

APO-BrdU TUNEL with Alexa Fluor 488

CellEvent Caspase-3/7 Green

MitoProbe DiOC2(3)

MitoProbe JC-1

YO-PRO-1 Iodide

CellTrace CFSE

Click-iT Plus EdU Alexa Fluor 488

CellROX Green

pHrodo Green E. coli BioParticles Conjugate

pHrodo Green S. aureus BioParticles Conjugate

EGFP

Emerald GFP

EYFP

BD Horizon BB515

574/26575/30580/23585/40586/15590/40

BL2

574/26 (without yellow laser present)

590/40 (with yellow

laser present)

PE

PE-Alexa Fluor 610

PE-Texas Red

PE-eFluor 610

LIVE/DEAD Fixable Red

7-AAD

Propidium Iodide (PI)

SYTOX AADvanced

SYTOX Orange

Vybrant DyeCycle Orange

FxCycle PI/RNase

Annexin V, PE

TMRE

TMRM

MitoProbe JC-1

pHrodo Red E. coli BioParticles Conjugate

pHrodo Red Phagocytosis Kit

EYFP

mCitrine

Venus

BD Horizon PE-CF594

PE/Dazzle 594

670/30675/30680/30695/30695/40710/50

BL3

695/40

PE-Alexa Fluor 700

PE-Cy®5.5

PerCP

PerCP-Cy®5.5

Qdot 705

TRI-COLOR (PE-Cy®5)

PerCP-eFluor 710

7-AAD

Propidium Iodide

SYTOX AADvanced

Annexin V, PerCP eFluor 710PerCP-Vio700

VioGreen

750 LP775/50780/40780/60785/70

BL4

780/60 (without yellow laser present)

PE-Cy®7

Qdot 800Vybrant DyeCycle

RubyAnnexin V, PerCP eFluor 710

PE-Vio770

VioGreen

Gree

n (5

32 n

m)

555/20575/24585/16585/42610/20

GL1

575/36

Alexa Fluor 532

Alexa Fluor 555

Qdot 565

Qdot 605

R-phycoerythrin (R-PE, PE)

SYTOX OrangeVybrant DyeCycle

Orange

Annexin V, R-PE


MitoTracker Orange CMTMRos

CellTrace Yellow CellROX OrangepHrodo Red E. coli

BioParticles Conjugate

DsRed

tdTomato

YFP

610/20620/15630/30

GL2

620/15

Alexa Fluor 568

Alexa Fluor 594

Qdot 605

PE-Texas Red


SYTOX AADvanced

Propidium IodideVybrant DyeCycle

OrangeFxCycle PI/RNase



MitoTracker Red CMXRos


mCherry PE/Dazzle 594

695/40710/50

GL3

695/40

Qdot 705

PE-Alexa Fluor 700

TRI-COLOR

PE-Cy®5.5

SYTOX AADvancedVybrant DyeCycle

Ruby

780/60GL4

780/60

Qdot 800

PE-Cy®7

Yello

w (5

61 n

m)

585/40585/15590/40

YL1

585/15

Alexa Fluor 555

PE

Propidium Iodide

SYTOX OrangeVybrant DyeCycle

Orange

MitoTracker Orange CMTMRos

MitoTracker Red CMXRos

TMRE

TMRM

CellTrace Yellow CellROX OrangepHrodo Red E. coli

BioParticles ConjugatemOrange

RFP

610/20613/20615/50620/15620/30

YL2

620/15

PE-Alexa Fluor 610

PE-Texas Red

PE-eFluor 610


7-AAD

Propidium Iodide

SYTOX AADvanced

SYTOX Orange

Vybrant DyeCycle Orange

FxCycle PI/RNaseAnnexin V, Alexa Fluor 568

Annexin V, Alexa Fluor 594Click-iT Plus EdU

Alexa Fluor 594

DsRed

mCherry

mKate

mStrawberry

tdTomato

ECD

BD Horizon PE-CF594

PE/Dazzle 594

PE-Vio 615

670/30675/30680/30695/30695/40

YL3

695/40

PE-Alexa Fluor 700

PE-Cy®5.5

Qdot 705

TRI-COLOR

7-AAD

Propidium Iodide

SYTOX AADvanced

mPlum

mRaspberry

mNeptune

750 LP775/50780/40780/60785/70

YL4

780/60

PE-Cy®7

Qdot 800Vybrant DyeCycle

RubyPE-Vio770

Red

(637

nm

)

660/20661/20665/20670/14670/30675/30

RL1

670/14

Alexa Fluor 647

APC

Qdot 655

eFluor 660

APC-Cy®5

LIVE/DEAD Fixable Far Red

SYTOX Red FxCycle Far Red



Annexin V, APC

MitoProbe DiIC1(5)

TO-PRO-3

CellTrace Far Red


CellROX Deep Red

720/30730/45

RL2

720/30

Alexa Fluor 680

Alexa Fluor 700

APC-Alexa Fluor 750

Qdot 705750 LP775/50780/40780/60785/70

RL3

780/60

APC-Alexa Fluor 750

APC-Cy®7

APC-eFluor 780

LIVE/DEAD Fixable Near-IR

Vybrant DyeCycle Ruby

BD APC-H7

APC-Vio770

300 nm 400 nm 500 nm 600 nm

IRUV

800 nm700 nm

To find out about Invitrogen™ antibodies, assays, and instruments for flow cytometry, go to thermofisher.com/flowcytometry

For Research Use Only. Not for use in diagnostic procedures. © 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. APO-BrdU is a trademark of Phoenix Flow Systems Inc. BD and BD Horizon are trademarks of Becton, Dickinson and Company. Cy is a registered trademark of GE Healthcare UK Ltd. Hoechst is a trademark of Hoechst GmbH. PE/Dazzle is a trademark of BioLegend Inc. Vio, VioBlue, VioGreen, Vio700, and Vio770 are trademarks of Miltenyi Biotec GmbH. COL03855 0517

The Invitrogen™ Attune™ NxT Flow Cytometer. For more information, go to thermofisher.com/attune

bioprobes 75 journal of cell biology applications · bioprobes 75. journal of cell biology...

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