cell proliferation and cell counting 06-23-09 · approaches to cell proliferation and cell...

Approaches to Cell Proliferation and Cell Counting.

Mike Ignatius, Ph.D.

Product Manager, Molecular Probes/Invitrogen

Ed Leber,

Technical Service Rep on e-Chat

Invitrogen Proprietary & Confidential

Technologies Invitrogen Offers.

• Cell Number by Metabolic Indicators– Vybrant Cell Metabolism Assay Kit– LIVE/DEAD Calcein AM Vitality Kits– Luminescent: ATP Determination Kit– Colorimetric: Alamar Blue, MTT

• Cell Number by DNA Content– CyQuant™, CyQuant™ NF Cell Division

by DNA synthesis– BrdU, antiBrdU– Click-iT™ EDU

• Cell Division by Tracer Dye Analysis– CFSE (CellTrace kits and reagent)– Vybrant™ DiI

• Cell Division by Cell Cycle Analysis– DyeCycle™ Dyes

• Other Solutions– Counting Beads.

• Countess, Automated Hemocytomer Like Cell Counting

Let me count the ways….


Covered Today

• Cell Number by Metabolic Indicators– Vybrant Cell Metabolism Assay Kit– LIVE/DEAD Calcein AM Vitality Kits– Luminescent: ATP Determination Kit– Colorimetric: Alamar Blue, MTT

• Cell Number by DNA Content– CyQuant™, CyQuant™ NF, CyQuant Direct™

Cell Division by DNA synthesis– BrdU, antiBrdU– Click-iT™ ( EDU

• Cell Division by Tracer Dye Analysis– CFSE (CellTrace kits and reagent)– Vybrant DiI

• Cell Division by Cell Cycle Analysis– DyeCycle Dyes

• Other Solutions– Counting Beads.

• Countess®, Automated Hemocytomer Like Cell Counting

Different assays

give different answers


Reagent summary for cytotoxicity or proliferation analysis

Extremely rapid

Easy Edu replaces difficult BrdU assays

Samples remain intact

Imaging, HCS, flow and microplate formats

No wash assay

Highly accurate, sensitive, and linear

Workflow choices

Easy, HTP, homogeneous assay

Economical

Non toxic to cells

Why choose:

Direct indicator of cell proliferation

Cell counting via DNA content to assess cytotoxic or proliferative effects independent of cellular metabolism

Cell viability and cytotoxicity, & indirect proliferation measurement

Primarily used for:

Which cells are actively proliferating?

Have my cells multiplied? How many cells are present?

What is the metabolic health of the cells?

Answers the question:

Measures rate of new DNA synthesis by EdU nucleoside analog incorporation into DNA with detection by copper-catalyzed “click” reaction

Quantitates relative number of cells in a population based on total DNA content by measuring DNA binding dye

Metabolic assay for cellular reducing environment where readout is proportional to number of living cells

How it works:

Click-iT™ EdU KitsCyQuant® AssaysalamarBlue®

Product


alamarBlue®


alamar Blue®- HTS Ready

• Measure of cell health thru cellular reducing potential• Live cells maintain reducing environment in cytosol• Excitation 540-570 nm / Emission 580-610 nm

– Monochrometer 560/590 nm• Simple add and read protocol• Proprietary reagent mix allows less volume addition and more dynamic range

Blue Red

resazurin resorufin

Healthy Cells

Dying/Dead Cells


alamarBlue®

How does the assay work? • Cellular metabolism converts alamarBlue® from

blue to a pink & fluorescent product that is released into media

• Healthy cells are more metabolically active than sick or dead cells :

– Healthy cells convert more reagent from blue to pink

Protocol in brief:

1. Add reagent to cells (10% v/v)

2. Incubate at 37 C for 15-30 min

3. Read results by absorbance or fluorescence in microplate reader

4. Returned to incubator and use from other assays e.g. reporter gene studies


alamarBlue® sensitivity

Sensitivity of alamarBlue® reagent after 18 hour incubation

The inset graph shows alamarBlue® to be linear over the range from 50 to 5,000 cells/well after 18 hour incubation of cells with reagent.


alamarBlue® gives nearly identical results at a fraction of the cost of CellTiter® Glo

Comparison of alamarBlue® reagent with CellTiter-Glo®. HUVEC cells were treated with tamoxifen for 24 hours prior to performing the cytotoxicity assays. The alamarBlue® (AB) and CellTiter-Glo® (CTG) assays were performed according to the manufacturer’s instructions.


alamarBlue® is more accurate in confluent cultures

alamarBlue® accurately shows no cell death in the confluent culture, whereas since ATP is depleted, CellTiter-Glo® indicates false cell death.

“Cells that become quiescent upon reaching confluency may have decreasing ATP levels and thus may not be accurately measured by the CellTiter® Glo assay at confluence.”

-0.50 -0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.500

5000

10000

15000

20000

25000

30000

35000

40000

45000

200000

250000

300000

350000

400000

450000

alamarBlue®

CellTiter-Glo®

log Tamoxifen (μM)

Bkg

Sub

RFU

Bkg Sub R

LU


CyQUANT® Kits

Most Sensitive and Robust Product Family for DNA-based Cell Proliferation and Cytotoxicity Studies


CyQUANT® Family of Cell Proliferation Kits

• Highly Sensitive: Much more sensitive than metabolically based assays to small changes in cell population/number (4 log dynamic range).

• Easy to use: Compatible with automation and high throughput analysis.

• Convenient: Choose from one of three kits to suit your assay and work flow needs.

• Accurate: DNA is tightly regulated and signal is not dependent on metabolic state, growth conditions—no better HTS assay for cell number

• Robust: Highly reproducible and not prone to compound interference—from assay to assay and lab to lab.

Nucleic acid-specific dye that fluoresces strongly green (ex=488 nm) when bound to DNA

Only DNA binding reagents suitable for HTS

100 platesC35012CyQUANT® Direct Cell Proliferation Assay Kit

2 platesC35007CyQUANT® NF Cell Proliferation Assay Kit

10 platesC35006CyQUANT® NF Cell Proliferation Assay Kit

10 platesC35012CyQUANT® Direct Cell Proliferation Assay Kit

10 platesC7026CyQUANT® Cell Proliferation Assay Kit

SizeSKUName


DNA Based Cell Number with CyQuant® Assays: Same Principle, Different Workflow Options.

First up, the original CyQUANT® assay (released in 1995)

1. Wash, Freeze, Add and Read. 2. Can be stored frozen for 4 weeks prior to read.3. Sensitive: from 50 to 250,000 cells4. Best in class dynamic range, linear over 4 logs5. Flexible: top- or bottom-read; 96 or 384 well formats.6. Ideal for time course studies (proliferation)

You can count on it!


CyQuant® NF: No Freeze, cells intact, HCS ready!

Second, the CyQuant® NF assay (released in 2005)

1. No freeze means antigenicity and cellular structures are preserved2. Multiplex with antibody- or dye-based assays in flow or HCS3. Use on fixed cells—no dependence on metabolic activity4. Simple protocol: Wash, Add and Read. Stable for hours.5. Best in class DNA based proliferation assay.

NF = No Freeze, New Formulation, Nice Format


Cyquant NF™ - Pharmacology

Normal Cells Toxicity DataTamoxifen 24 Hrs

Cyquant NF

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.251,000,000

3,000,000

5,000,000

7,000,000

9,000,000

11,000,000

13,000,000

IC50SH-SY5Y-59.2 μM

Tamoxifen (log M)

Bkg

Sub

RFU

Primary Cells Toxicity DataTamoxifen 24 Hrs

Cyquant NF

-0.50 -0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.500

500,000

1,000,000

1,500,000

2,000,000

IC50HUVEC- 6.3 μM

Tamoxifen (log M)

Bkg

Sub

RFU

•Pharmacological findings match those seen with alamar Blue®•Primary Cells more sensitive to drug effects as seen with AB

•Unlike alamarBlue® a media removal step is required•Haven’t yet tested with New CyQuant™ DIRECT.•Mechanistic readout is not based on cellular metabolism


alamarBlue® and CyQUANT® can be used together

HepG2 cells were incubated with tamoxifen for 24 hours prior to the addition of reagnets. The indicated IC50 values demonstrate that alamarBlue®reagent can be multiplexed with other cytotoxic indicators and still give accurate results.

Potential multiplexing advantages

•Save on resources

•Get a more accurate indication of cell health due to the multiple assessment modes used

A) alamarBlue® reagent

B) CyQUANT® NF

C) alamarBlue® reagent and CyQUANT® NF


Now: CyQUANT® Direct Cell Proliferation Assay

Quantitative, sensitive, and non-radioactive indicator for cell cytotoxicity and proliferation

Dye turns fluorescent upon binding to DNA

Normal Cells Toxicity DataTamoxifen 24 Hrs

Cyquant NF

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.251,000,000

3,000,000

5,000,000

7,000,000

9,000,000

11,000,000

13,000,000

IC50SH-SY5Y-59.2 μM

Tamoxifen (log M)

Bkg

Sub

RFU

Cells + Drug candidate

Add CyQUANT® reagent Measure

What is it?

Homogenous version of CyQUANT® dye, combined with a masking dye that prevents staining of cells with damaged membrane

Why use it?

• Convenient workflow – one addition, fast labeling, no washes, cell lysis or temperature equilibrations required. Room temp storage.

• Reported to be among the most sensitive measures of cytotoxicity and the most accurate assay for cell number (proliferation)

• Readout independent of metabolic state gives excellent robustness and may reduce the risk for false positives

• Highly robust and accurate

New!!!


CyQUANT® Direct: Add and Read Protocol

30 to 60 minTreatment Signal from stained dead & damaged

cells blocked by masking dye

– Homogenous assay with fast, work flow-friendly protocol

– Only healthy cells are stained—two assays in one (DNA and membrane integrity)

– Metabolic activity is not needed for staining (e.g. confluent, quiescent cells)

– Can be used with either adherent or suspension cells (bottom read recommended)


CyQUANT® Direct: Fast Labeling

Fast labeling kinetics


CyQUANT® Direct: Visualize the Results!

PrimaryHASMC Jurkat

Hep G2 CHO

Inspect cell morphology, confluence, cell numbers visuallyMultiplex with spectrally distinct fluorescent markers or

luminescence readoutInspect by microscope and analyze by HCS


Choose CyQUANT® Kits Based on Application/Workflow

CyQUANT® NF Cell Proliferation Assay Kit1. No freeze, includes permeabilization step2. Antigenicity and cell structure preserved.3. Recommended for flow, HCS, imaging and assays using antibodies.

CyQUANT® Cell Proliferation Assay Kit1. Requires freeze step, but most sensitive2. Store frozen for weeks prior to read3. Linear over 4 logs from 20 to 50,000 cells4. Recommended for time course, batch assays and top-read instruments

CyQUANT® Direct Cell Proliferation Assay Kit1. Homogenous by combo of masking dye and DNA stain—add, mix, read 2. Fast and convenient—room temp storage, no lysis, no freeze, no perm 3. Most robust, accurate and stable signal4. Measures DNA content and membrane integrity (proliferation and

cytotoxicity)5. Recommended for high throughput applications in bottom read mode


Viability AssaysLive Dead, Cytotoxicity Assay.In live cells, esterase converts Calcein AM from non-fluorescent to green. Dead cells accumulate the another dye to stain red.

•Red Dead, Green Growing.


Viability & Vitality: LIVE/DEAD® Kits

No488nmPropidium IodideSYBR® 14 dyeLIVE/DEAD® Sperm Viability

KitL7011

Yes488nmGreen Dye

brightGreen Dye

dimLIVE/DEAD® Reduced

Biohazard Cell Viability Kit #4L23105

Yes488nmRed Dyebright

Red Dyedim

LIVE/DEAD® Reduced Biohazard Cell Viability Kit #3L23102

YesUVBlue Dyebright

Blue Dyedim

LIVE/DEAD® Reduced Biohazard Cell Viability Kit #2L23101

Yes488nmReactive RedSYTO® 10 dyeLIVE/DEAD® Reduced

Biohazard Cell Viability Kit #1L7013

No488nmPropidium IodideDiOC18(3)LIVE/DEAD® Cell-

Mediated/Cytotoxicity KitL7010

No488nmEthidium Homodimer-

1Calcein AM

LIVE/DEAD® Viability/ Cytotoxicity Kit

L3224

Fix?ExcitationDead StainLive StainKit NameSku #

LIVE/DEAD® lineup: pick your colors


Click-iT™ Labeling and Detection

Replacing radioactivity with fluorescence for sugars, amino acids and nucleic acid analysis in living cells.

Novel tools for fluorescence imaging, gel electrophoresis, and flow cytometry - AND CELL PROLIFERATION – BRDU REPLACEMENT


Concept introduce by K. Barry Sharpless in 2001. Has become associated in particular with one reaction.

Kolb HC, Finn MG, Sharpless K.B.; (2001) Angewandte Chemie International Edition V40:2004-2021.

Tornoe CW, Christensen C, Meldal M; (2002) J Org Chem. May 3;67(9):3057-64.

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)

•Highly efficient•Rapid•Bio-orthogonal•Components are inert to biomolecules•No side reactions

DetectionProbe N3 Macromolecule

N NN

R''

R'

Macromolecule

DetectionProbe

Copper I

Just what is “click” chemistry?

+


•Metabolic•Enzymatic•Chemical

Phosphoproteins

Glycoproteins

Total proteins

DNA/RNA

Lipids

Incorporate azide/alkyne tag into macromolecule of interest

•1D-2D gel imaging

•Western blotting

•Fluorescence microscopy

•Flow cytometry

•Enrichment

•In situ hybridization

•Fluorescent•Biotinylated•Affinity tags

Click-iT label macromolecule with desired azide/alkyne probes

The Click-iT™ 2-step labeling and detection approach

•REPLACEMENT FOR BrDU for PROLIFERATION


Tritiated (3H) thymidine

TRADITIONALLY1960’s - (3H-thymidine)


Br

Br

Br

Br1970’s - (BrdU)


Acid or DNase

Br

Br

Br

Br

BrdU - is inaccessible to the BrdU antibody

Requires DNA denaturation for strand separation

Cell cycle dye requires the DNA to be double-stranded

Difficult balancing act:

Numerous protocols: acid, heat, or nuclease for DNA denaturation


Br

Br

Br

Br


Ethynyl dU Bromo dU

H

Nucleoside analogue structures

Click-analogue


Chemistry

Click labeling doesn’trequire DNAdenaturation

Dye azide reacts with the alkyne on the double stranded DNA


Click-iT™ EdU Protocol

TOTAL TIME70 minutes

Image

15 minutesWash 3X

15 minutesNuclear counterstain

10 minutesWash 2X

30 minutesClick-iT™ detection reaction

Incubate with EdU or BrdU, fix & permeabilize sample

With Click-iT™ EdU

• Measure proliferation in cells or tissue

• Time to complete: <2 hours

• Detect by fluorescence microscopy, flow cytometry or high-throughput imaging (HCS)

BrdU Protocol

15 minutesNuclear counterstain

15 minutesWash 3X

15 minutesWash 3X

15 minutesWash 3X

60 minutesBlock

1-16 hoursAnti-BrdU incubation

120 minutesSecondary antibody incubation

TOTAL TIME 6-21 hours

Image

15 minutesWash 3X

12 minutesNeutralize

40 minutesHCl Denaturation

15 minutesWash 3X

Click-iT™ EdU vs. BrdU Workflow


Click-iT™ EdU vs BrdU Detection in Tissue Sections

•ClickiT™ or Ticket…


Click-iT™ EdU in Mouse Intestine.


Click-iT™ EdU in Adult Brain Tissue.

Adult Mouse brain section, an organ whose cells almost never divide.

A sole EdU-labeled cell can be easily identified on this brain section.

Courtesy of Adrian Salic, Harvard.

Research published in

Feb 2008, PNAS 105(7) 2415-2420.


Muntjac cells pulsed with 10 μM EdU for 1 hour

Multicolor imaging with Click-iT™ EdU. Newly synthesized DNA with Click-iT™EdU (pink), tubulin (blue), golgi (green) and peroxisomes (orange).

EdUtubulingolgiperoxisome


Cell cycleMitosis

S = period of EdU uptake

G0G1

G0: quiescent cells

G1: Period of cell growth where a cell has 2N nuclear DNA content (2 copies of nuclear genome)

S: Period where nuclear DNA content doubles to 4N EdU Uptake Occurs here.

G2: Period of cell growth where the cells are maintained at 4N

M: Cell division resulting in two daughter cells each with 2N nuclear DNA content

G2


Click-iT™ EdU cell proliferation : Flow Cytometry

7-AAD fluorescence

New azide

labeled with

Alexa Fluor® 488 dye

Traditional 7-AAD

cell cycle dye

Hela cells:

Human cervical carcinoma cells


Measure S phase entry with Click-iT™ EdU. HCS duplicates Flow data!

• Images: HeLa cells, EdU labeling for 120 min.; formaldehyde fixed and processed for Click-iTTMEdU reaction; DNA -Hoechst 33342; EdU –Click-iTTM Alexa Fluor®488 Azide Dye.

• Histograms show relative frequencies; “EdU positive” is green (bottom right); dynamically linked graph of DNA profile (bottom left) shows the distribution of “EdU positive” in DNA profile.


Click-iT™ EdU Kits

A102082 plate (~200 tests)

Click-iT™ EdU Alexa Fluor® 647 High-throughput Imaging (HCS) Assay Kit

2 plate

(~200 tests)

2 plate

(~200 tests)

50

50

50

Number Tests

A10202Click-iT™ EdU Alexa Fluor® 647 Flow Cytometry Assay

A10034Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay

A10044EdU (5-ethynyl-2’-deoxyuridine)

A10209Click-iT™ EdU Alexa Fluor® 594 High-throughput Imaging (HCS) Assay Kit

A10027Click-iT™ EdU Alexa Fluor® 488 High-throughput Imaging (HCS) Assay Kit

C35002Click-iT™ EdU Alexa Fluor® 488 Flow Cytometry Assay

Catalog NumberProduct

Countess™ automated cell counter


Countess™ automated cell counter

NEVER USE A HEMOCYTOMETER AGAIN!

Accurately counts cells in 30 seconds

Calculates % viability

Measures average cell size

Includes dilution calculator

Call for a demo today

Automated cell counting at your fingertips


Countess is a complete counting platform

C10227 Countess automated cell counter)– Includes Countess instrument, box of 50 slides and trypan blue

C10228 Countess™ cell counting chamber slides, 50– Includes trypan blue

*C10288 Countess™ field test kit (for internal sales only)– Includes box of 50 slides, trypan blue, beads and instructions


Countess™ makes cell counting easy

3. Focus and Press “Count cells”

2. Insert slide into Countess instrument

1. Mix 10 uL of sample with 10 uL of trypan blue & pipet into Countess chamber slide


Focus on cells before counting


Countess Software – Look at cell count


Countess Software – Report Printout


Countess data has a wider range than hemocytometer

1.00E+05

1.00E+06

1.00E+07

1.00E+05 1.00E+06 1.00E+07

Cell suspension dilution

Mea

sure

d co

ncen

tratio

n

Countess™ automatedcell counterHemocytometer

Countess Range1x104 – 1x107 cells/mL

– Highest accuracy: – 1x105 – 4x106 cells/mL

Hemocytometer range– 2.5x105 – 2.5 x106

cells/mL


The Countess works with many cell types

Cell type Species Tissue Primary or Immortalized Cell line Average cell size

293-Herg Human kidney immortalized 13 umA431 Human skin immortalized 15.5 umCHO-M1 Hamster ovary immortalizedCHSE Fish embryonic 16-17 umCOLO-207 Human intestine immortalizedCOS-7 Monkey kidney immortalizedHASMC human smooth muscle primary 20 umHeLa Human cervix immortalizedHepG2 Human liver immortalized 18 umHL-60 Human blood immortalizedHPAEC human artery endothelial primary 13 umHPASMC Human smooth muscle primary 20 umHUVEC Human umbilical vein primary 17 umJ774(MMM) Mouse blood immortalized 13-14 umJurkat Human blood immortalized 12 umK-562 Human bone/marrow immortalizedMCF-7 Human mammary immortalized 20-24 umMRC-5 Human lung immortalized 18 umNIH/3T3 Mouse embryonic immortalized 18 umPC-12 Rat adrenal immortalized 12-14 umSF-21 insect ovaryU266 Human blood immortalized 12-13 umU2OS. Human bone/marrow immortalizedAdipocytes human fat primary 16-17 ummelanocytes Human skin primarykeratinocytes Human skin primary

Counts cell sizes 5 – 60 microns

Concentration range is 1 x 105 – 4 x 106

cells/mL

Does it count clumpy cells?

Yes, it can count clumps of 2-5 cells, but

not larger clumps


The Countess makes your science better

BETTER ACCURACY Minimize subjective counting. Can easily count more cells to increase accuracy

BETTER DOWNSTREAM RESULTS You are more likely to count when you “should,”rather than skip this step

INCREASE YOUR VERSATILITY Broadens capabilities for experimental design –easy to count many samples

FASTER COUNTING Takes 30 minutes to count and archive 30 samples

BIOHAZARD ISSUES Disposable slides better for biohazardous materials


THANK YOU!

Questions? [email protected]

800.955.6288

cell proliferation and cell counting 06-23-09 · approaches to cell proliferation and cell...

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