guava easycyte systems - dev.luminexcorp.com...guava ® easycyte systems e tometry unleash what’s...

GUAVA® EASYCYTE SYSTEMS Expanding the potential of flow cytometry

The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.

2

Guava® easyCyte Systems │ Expanding the potential of flow cytometry

UNLEASH WHAT’S POSSIBLEOver fifteen years ago, Guava Technologies introduced the first compact benchtop flow cytometers. Today, the Guava® easyCyte line has been updated to offer up to 3 lasers and 14 parameters with greater sensitivity and optional high throughput capabilities. Powered by intuitive software, the Guava® easyCyte flow cytometers are some of the most dynamic and flexible benchtop systems available.

• Up to 3 lasers and 14 parameters on a benchtop instrument

• Microcapillary fluidics design eliminates sheath fluid and waste carboys

• Accurate absolute cell counts via microcapillary and positive displacement syringe pump

• Intuitive software includes comprehensive cell-health related assays

• Detection of particles as small as 0.2 μm

• High-throughput option for walk-away acquisition of up to 96 samples

Sheath Fluid: None

Waste

Laser

Flow

Cel

l

Sample

Sam

ple

Flow

Waste: < 50 mL/8 hour run

Typical # Cells:10,000 - 1,000,000 cells/mL

Microcapillary Flow Cytometry

3

Inside the Guava® easyCyte 12HT systems

405

nmNIR

785/

70RE

D69

5/50

RED

-R66

1/15

YLW

583/

26G

RN52

5/30

488

nm

642

nmSSC

FSC

BLU448/50

How it Works

The Guava® easyCyte systems use patented, microcapillary, laser-based technology capable of detecting mammalian and microbial cells, particles, and beads. A sample of fluorescently labeled cells is aspirated into a uniquely proportioned microcapillary flow cell. Forward and side scatter characteristics are detected by photodiode, and fluorophores excited by the violet, blue, or red laser emit signals that are spectrally filtered to resolve up to 10 fluorophores simultaneously.

405 nm - Violet Laser

488 nm - Blue Laser

642 nm - Red Laser

Blu - V GRN - V YEL - V RED - V

GRN - B YEL - B RED - B NIR - B

RED - R NIR - R

(448/50)BV421™

Cascade Blue

(525/30)BV520™

Cascade Green

(583/26)BV605™

(695/50)BV650™

(525/30)FITC

AF® 488(583/26)

PE(695/50)PE-Cy5.5

(785/70)PE-Cy7

(661/15)APCCy5

(785/70)APC-Cy7

APC-AF® 750

4


Sensitive and specificSpherotech 8-color beads analyzed on the Guava® easyCyte 12HT system demonstrate the instrument’s proficiency for resolving adjacent fluorophores in multiple detection channels.

100

140 Cascade Blue MESF: 40Plot P02, gated on P01.R1

Blue - V Fluores (BLU-V-ALog)

100

70

40

10

101 102 103 104 105

Count

100

140PE-Cy5 MESF: 120Plot P05, gated on P01R1

Red - B Fluores (Red-B-ALog)

100

70

40

10

101 102 103 104 105

Cou

ntBlue Fluorescence - 405nm laser Red Fluorescence - 488nm laser

100

140PE-Cy7 MESF: 120Plot P06, gated on P01R1

Near IR-B Fluo...a (NIR-B-ALog)

100

70

40

10

101 102 103 104 105

Cou

nt

100

140APC MESF: 50Plot P07, ungated

RED-R Fluores... (RED-R-ALog)

100

70

40

10

101 102 103 104 105

Cou

nt

NIR Fluorescence - 488nm laser Red Fluorescence - 642nm laser

100

140 FITC MESF: 25Plot P04, gated on P01. R1

Yellow-B Fluor...a (YEL-B-ALog)

100

Cou

nt

70

40

10

101 102 103 104 105 100

140 FITC MESF: 75Plot P03, gated on P01. R1

Green-B Fluor... (GRN-B-ALog)

100

70

40

10

101 102 103 104 105

Cou

nt

Yellow Fluorescence - 488nm laser Green Fluorescence - 488nm laser

5

Plot P04, gated on PO1.CD45+.CD3-.R9

100

101

10

2

1

03

104

CD

19 B

V 5

10 (

GR

N-V

-HLo

g)

CD19+

CD16&CD56+

101 102 103

CD16 + CD56 PE (YEL-B-HLog)

10 μL adult human blood was stained for 20 minutes at room temperature with a cocktail containing anti-CD45 PerCP-Cy5.5, anti-CD3 Brilliant™ Violet 421, anti-CD4 PE-Cy7, anti-CD8 APC-Cy7, anti-CD16+CD56 PE, anti-CD19 Brilliant™ Violet 510, anti-CD45 RA APC, and anti-CD62L FITC. After incubation, cells were lysed and fixed with 180 μL Guava® lysing solution for 15 minutes at room temperature. Samples were then acquired on the Guava® easyCyte 12HT system. Lymphocytes identified as CD45+ were selected and subsequently gated into a SSC vs. CD3 plot. T cells (CD3+ and CD45+) were gated into a CD4 vs CD8 plot. CD4+ and CD8 + T cells were subtyped by evaluating each population using CD45RA and CD62L to differentiate naive from memory cells. To distinguish natural killer (NK) and B cells, CD3-negative cells were gated into a plot comparing CD19 (B cells) and CD16+56 (NK cells).

Immunological Phenotyping

Plot P01, ungated

101 102 103

0 1

0000

3

0000

5000

0

700

00

90

000

CD45 PerCP-Cy5.5 (RED-HLog)

CD45+

Sid

e Sca

tter

(SSC

-HLi

n)

Plot P02, gated on P01.CD45+

101 102 103

0 1

0000

3

0000

5000

0

700

00

90

000

CD3 BV 421 (BLU-V-HLog)

Sid

e Sca

tter

(SSC

-HLi

n)

CD3- CD3+Plot P03, gated on PO1.CD45+.CD3+

101 102 103 104

CD8 APC-Cy7 (NIR-R-HLog)

101

10

2

103

10

4

CD

4 PE-C

y7 (

NIR

-B-H

Log)

CD4+

CD8+

Plot P05, gated on P01.CD45+.CD3+.CD

101 102 103 104

CD45RA APC (RED-R-HLog)

CD

62L

FITC

(G

RN

-B-H

Log)

100

10

1

102

1

03

104

R6

Plot P06, gated on P01.CD45+.CD3+.CD

101 102 103 104

CD45RA APC (RED-R-HLog)

CD

62L

FITC

(G

RN

-B-H

Log)

100

101

10

2

1

03

104

R6

CD45+

CD3+

CD3-

CD4+

CD8+

6


1. Create and apply analysis methods across multiple dat asets.

2. Perform compensation during acquisition or analysis or use the automated compensation features.

3. Drag-and-drop gating.

4. View up to 24 plots at once.

5. Refine gates in real time.

6. Default or custom statistics.

7. Multiple plot and gating options.

8. Minimal gain adjustment needed when performing routine assays.

9. Analyze both tubes and plates.

SoftwareThe guavaSoft® operating system software provides access to modules for acquisition and analysis, as well as instrument setup and maintenance. The guavaSoft® system includes templates for use with a wide range of MilliporeSigma flow cytometry kits to simplify your experiments and data collection. Additionally, the guavaSoft® package includes InCyte™, an intuitive open software package for custom analysis. Results can be exported to spreadsheets or as industry-standard FCS 2.0 or 3.0 files for further analysis. GuavaSoft® software includes 21 CFR Part 11-enabling features.

InCyte™ Software: IntuitiveOur InCyte™ software has an intuitive, easy-to-use interface that enables you to focus on data at the sample or experimental level. The software simplifies setup and analysis of plots with drag-and-drop features, while automated compensation makes it easy to perform complex, multi-color assays. The instant update feature responds in real time to change analysis conditions for viewing. The multiparameter heat mapping function allows analysis of entire plates of data in the time previously required to analyze a single sample. These features provide a simple and rapid means to attain a macroscopic view of experiment “hits” and easily compare different experiments in real time. InCyte™ software is especially useful for interpreting the results of high-throughput cell-based assays.

Annexin V CF400 (GRN - HLog

7AAD

(RED

- H

Log)

Plot PO3, gated on P01.R1

Mito

Sens

e Re

d (R

ED2

- H

Log)

Annexin V CF488 (GRN - HLog

Plot PO2, gated on P01.R1Plot PO1, ungated

Forward Scatter (FSC - HLog)

Side

Sca

tter

(SSC

- H

Lin)

1 2 3 4 5 6 7 8 9 10 11 12

Count: 2746

100 101 102 103 104020

0040

0060

0080

00

A

B

C

D

E

F

G

H

MitoSense Red (+)Annexin V (-)68.46%

MitoSense Red (+)Annexin V (+)

4.44%

MitoSense Red (-)Annexin V (-)6.19%

MitoSense Red (-)Annexin V (+)

20.90%100 101 102 103 104

100

10

1

102

10

3

104

100

10

1

102

10

3

104

100 101 102 103 104

2 3 4 5

6

7

8

9

1

7

0 1

0 2

0 3

0 4

0 5

0 6

0 7

0 8

0 9

0

11

0

IC50=4.9µM

HeLa 24 hours

IC50 determination using the Cytochrome c Kit was analyzed with the built-in IC50/EC50 curve fitting feature of InCyte™ software. Cells were acquired on the Guava® easyCyte 8HT system. Plot A shows the drag-and-drop gating strategy used for the IC50

A.

Res

pon

se L

evel

, %

Res

pon

se L

evel

, %

0 1

0 2

0 3

0 4

0 5

0 6

0 7

0 8

0 9

0

11

0

10-1 100 101 102 103 10-1 100 101 102 103

Concentration Concentration

IC50=38µM

B. C.

HeLa cells in microtiter plates were treated with various cytotoxic compounds for 24 hours. Cells were stained using MilliporeSigma’s MitoDamage, MitoCaspase, or MitoStress kits. Cells were acquired on the Guava® easyCyte system and percent population data were compared in a heat map format using our InCyte™

InCyte™ Software Heat Map View

IC50 Determination within InCyte™ Softwaredetermination. Plot B shows the IC50 curve results for gambogic acid and Plot C shows the IC50 for etoposide. The once-complex task of generating the IC50 or EC50 curve for a given compound is automated by InCyte™ based on quantitation of fluorescent signal.

Software. The InCyte™ heat map function facilitated the rapid identification of compounds inducing positive results, by comparison of all 5 parameters simultaneously as shown in the pie charts above. The data show the results for cells treated with 80 different compounds in a single plate.

Combine groups of data to construct heat maps, IC50, or EC50 curves.

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

0 -20%

20 -40%

40 -60%

60 -80%

80 -100%

MitoSense Red

MitoStress

Annexin V-CF488

Caspase 3/7

7-AAD

8


Small Particle DetectionThe Guava® easyCyte 8HT and 12HT systems have been shown to detect particles as small as 0.2 µm, a significant improvement over typical flow cytometers. This increased resolution and sensitivity means better separation, making gating and identification of dim populations easier. These capabilities may prove particularly useful for researchers analyzing particulates, beads, bacteria or algae.

Acquisition of a mixture of beads of known size demonstrates the ability of Guava® easyCyte 12HT instruments to detect and discriminate particles as small as 0.2 µm.

Lipid measurement of chlorophyll A-positive algae. Identification of algal cells containing chlorophyll A; chlorophyll A fluoresces in the red channel (A). Gate applied to select for chlorophyll A-positive cells (B). Histograms showing a wide range of lipid content (as evidenced by BODIPY green fluorescence intensity) for a variety of algal strains (C), with one clone showing as much as 500 times the lipid content as others.

Turning algae into biofuelsGuava® easyCyte systems are currently participating in algal biomass laboratories worldwide, where flow cytometry facilitates selection of high lipid content strains and efficient monitoring of cultures. Because microcapillary systems require smaller sample volumes, generate significantly less waste, have lower operating costs, enable high sample throughput, and have a small instrument footprint, they are a natural choice for demanding laboratory settings.

A. Not Gated B. Gated by Chlorophyll A

C. Histograms showing varying lipid content of different algae clones

Sid

e-S

catt

er (

SS

C-H

Log)

Forward Scatter (FSC-HLog)

1.3µm

0.8µm

0.4µm0.2µm

100

100 101 102 103 104 105

101

102

103

104

105

100 101 102 103 104

100

101

102

102

104

Chlorophyll A (RED-HLog)

Sid

e Sca

tter

(SSC

-HLo

g)

100 101 102 103 104

0

38

75

113

150

BODIPY (GRN-HLog)

Cou

nt

M1 M2

100 101 102 103 104

0

5

10

20

30

BODIPY Fluorescence

Cou

nt

High LipidContent Clone

9

Mouse peripheral blood cells gated on lymphocytes (left) were stained with Milli-Mark® FCMAB244CP5 anti-mouse CD4 (clone GK1.5) PerCP/Cy5.5. CD4+Th1 cells were identified (right) by costaining with FCMAB243P, anti-mouse IFN gamma (clone XMG1.2) PE. Data were analyzed with InCyte™ software on a Guava® easyCyte HT cytometer.

Uninduced

MitoS

ense

Red

MitoS

ense

Red

Annexin V, CF488A

7-AAD

Red

2 Fl

uore

sece

nce

(R

D2-

HLo

g)Red

2 Fl

uore

sece

nce

(R

D2-

HLo

g)

Green Fluorescence (GRN-HLog)

Red Fluorescence (RED-HLog)

Green Fluorescence (GRN-HLog)

Red Fluorescence (GRN-HLog)

94.4%

0.75% 3.7%

1.1%

100

100

100

100

101

101

101

101

102

102

102

102

103

103

103

103

104

104

104

104

100

100

100

100

101

101

101

101

102

102

102

102

103

103

103

103

104

104

104

104

54.7%

14.6% 27.0%

3.7%

2 µM Staurosporine

95.2%

3.2% 1.3%

0.3%

Depolarized Cells

Dead Cells

Live Cells58.1% 0.08%

41.0% 0.8%

Plot P02, gated on P01.Live

101 102 103 104

CD4-PerCP (RED-HLog)

IFN

g-P

E (

YEL-

HLo

g)

100

101

102

103

104

Plot P03, gated on P04.R1.R2

101 102 103 104

0 10

000

30

000

500

00 7

0000

9

0000

CD4 (RED-HLog)

Sid

e Sca

tter

(SSC

-HLi

n)

FlowCellect® Flow Cytometry Kits MilliporeSigma’s optimized, turnkey assay kits reduce sample preparation time, minimize assay development and simplify data analysis.

We offer more than 40 FlowCellect® kits optimized for key assays in cell health, immunology and cell signaling.

The FlowCellect® MitoDamage Kit for flow cytometry contains MitoSense Red, a fluorescent cationic dye that accumulates in the mitochondria and is responsive to changes in mitochondrial potential, a hallmark of early apoptosis.

Flow Cytometry Reagents

Fluorescence-conjugated Flow Cytometry AntibodiesChoose from our growing portfolio of over 1500 conjugated antibodies, including antibodies specific to many CD markers and immune signaling targets. With the reliability and quality of Upstate, Chemicon and Calbiochem, MilliporeSigma’s conjugated antibodies validated for flow cytometry are available in multiple colors, to enable you to unequivocally discriminate cell subpopulations:

All of our flow-validated antibodies come with our 100% performance and satisfaction guarantee.

Our diverse portfolio of reagents and assays facilitate fluorescence-based detection of proteins and nucleic acids, and have been validated for use on the Guava® easyCyte instrument platform.

Dot plots depicting Jurkat cells treated with multiple inducers and stained using MitoDamage kit. Dot plots of cells treated or not with staurosporine to induce apoptosis and acquired on the Guava® easyCyte 12HT system show percentages of cells positive for MitoSense Red (left). The guavaSoft® MitoDamage module facilitates experiment-level assessment of changes in mitochondrial potential that signal early apoptosis via heat mapping of up to 96 samples.

• FITC

• APC

• PE-Cy7

• PerCP-Cy5.5

• PE

• PE-Cy5

• APC-Cy7

• Violet450

10


Guava® easyCyte Single Sample System 5 5 HPL 6-2L 8

Catalog No. 0500-5005 0500-5009 0500-5007 0500-5008

Violet (405 nm) Laser

Blue (488 nm) 50 mW Laser


Green (532 nm) Laser

Red (642 nm) Laser

FSC

SSC

Blue-V (450/45 nm)

Green-V (512/18 nm)

Green-V (525/30 nm)

Yellow-V (575/25 nm)


Orange-V (620/52 nm)

Red-V (695/50 nm)

NIR-V (785/70 nm)

Green-B (512/18 nm)

Green-B (525/30 nm)

Yellow-B (575/25 nm)


Red-B (695/50 nm)

NIR-B (785/70 nm)

Yellow-G (575/25 nm)

Orange-G (609/30 nm)


Red-G (695/50 nm)

NIR-G (785/70 nm)

Red-R (664/20 nm)

NIR-R (785/70 nm)

Microcapillary Fluidics

Direct, Absolute Cell Counts

Automation-plate and tubes

Mixing

Dell® Laptop

InCyte™ Software

Digital Signal Processing

Guava® easyCyte Single Sample Systems

11

Guava® easyCyte HT Systems

Guava® easyCyte HT System 5HT 5HT HPL 6HT-2L 8HT 11HT 12HT BG HT BGR HT BGV HT

Catalog No. 0500-4005 0500- 4009 0500-4007 0500-4008 0500- 4020 0500-4012 0500-4015

0500-4025 0500-4030

Violet (405 nm) Laser



Green (532 nm) Laser

Red (642 nm) Laser

FSC

SSC

Blue-V (450/45 nm)

Green-V (512/18 nm)

Green-V (525/30 nm)



Orange-V (620/52 nm)

Red-V (695/50 nm)

NIR-V (785/70 nm)

Green-B (512/18 nm)

Green-B (525/30 nm)



Red-B (695/50 nm)

NIR-B (785/70 nm)

Yellow-G (575/25 nm)



Red-G (695/50 nm)

NIR-G (785/70 nm)

Red-R (664/20 nm)

NIR-R (785/70 nm)

Microcapillary Fluidics

Direct, Absolute Cell Counts

Automation-plate and tubes

Mixing

Dell® Laptop

InCyte™ Software

Digital Signal Processing

Description Cat. No.

Single Sampling Instruments

Guava® easyCyte 5 Base System 0500-5005

Guava® easyCyte 5HPL Base System 0500- 5009

Guava® easyCyte 6-2L Base System 0500-5007

Guava® easyCyte 8 Base System 0500-5008

High Throughput Sampling Instruments

Guava® easyCyte 5HT Base System 0500-4005

Guava® easyCyte 5HT HPL Base System 0500- 4009

Guava® easyCyte 6HT-2L Base System 0500-4007


Guava® easyCyte HT BG Base System 0500-4015


Guava® easyCyte HT BGR Base System 0500-4025


Guava® easyCyte HT BGV Base System 0500-4030

Ordering Information

Ver. 1.02018 - 09556

04/2018

© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. MilliporeSigma, the vibrant M, FlowCellect, Guava, InCyte and guavaSoft are registered trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.

MilliporeSigma400 Summit DriveBurlington, MA 01803

To place an order or receive technical assistanceIn the U.S. and Canada, call toll-free 1-800-645-5476 For other countries across Europe and the world, please visit: EMDMillipore.com/offices For Technical Service, please visit: EMDMillipore.com/techservice

EMDMillipore.com

http://www.emdmillipore.com/offices

http://www.emdmillipore.com/techservice

http://www.emdmillipore.com

guava easycyte systems - dev.luminexcorp.com...guava ® easycyte systems e tometry unleash what’s...

Documents