Data SheetData Sheet

Guava® ViaCount® Assay Rapid, accurate cell counting and viability assessments

The Guava ViaCount Assay is fast becoming the new

standard for viability and cell counting. In this simple

no-wash, mix-and-read-assay, you can accurately

obtain absolute total cell counts, perform viability

assessments, and determine apoptotic percentages—

all from tiny samples.

You’ll enjoy several advantages over traditional

methods, including greater accuracy, reproducibility,

and speed. This assay eliminates the user-to-user

variability characteristic of trypan blue hemocytometer

counting. As a result, you have significant increases in

counting reproducibility. In addition, the Guava ViaCount

Assay is more accurate than hemocytometer counting

or impedance-based counting over a wide range of

cell concentrations. Cell counting with this assay has

also proven to be up to 10 times faster than manual

hemocytometer methods. Unlike traditional cytometer

counting, you don’t need to use expensive reference

beads because of Guava’s unique capillary technology.

Cell counting and viability assessments can be used

in a variety of applications, such as cytotoxicity studies,

PBMC counting, rapid apoptosis assessment, and

increasing reproducibility in cell-based assays.

FeaturesAssay:

• Simple no-wash, mix-and-read procedure

• Analyzes viability of individual cells

• Delivers direct, absolute cell counts and population

percentage

• Counts up to 10 times faster than manual methods

• More reproducible than traditional tests

Samples: • Uses small samples in tubes or a 96-well plate

• Handles low-density and small-volume cell samples

• Works with adherent or suspended cells and mammalian

and insect cells

Compatibility: • Runs on Guava EasyCyte™ Mini, EasyCyte Plus, PCA,

and PCA-96 Systems

Debris Viable Cells Apoptotic Dead Cells

Forward Low High High HighScatter

Nuclear Neg High High HighStain

Viability Neg Neg Med HighStain

2

Mean

1 stdv

2 stdv

3 stdv

1200

800

600

2000

1800

1400

1000

1600

cel

ls6

SFC

/10

Assay Number

1 2 3 4 5 6 7 8

Manual via Trypan Blue

Guava ViaCount

Figure 3. Increased reproducibility in ELISPOT assays when using

Guava ViaCount assay for cell counting.

Figure 1. Guava ViaCount assay provides accurate cell counts.

Comparisons with an impedance counter and manual trypan blue

hemocytometer counting indicated greater linearity and accuracy over a

range of cell concentrations. Expected cell counts were determined using

an FDA-cleared reference counting bead method 1.

cel

ls/m

L) 8

6

6O

bser

ved

Cou

nt (x

10

4

2

0

Expected Count (x106 cells/mL)

0 2 4 6

Hemacytometer Impedance Countery=0.95x + 0.23 y=1.27x + 0.17R2=0.877 R2=0.964

Guava PCAy=0.94x + 0.25R2=0.966

Guava PCA ( )

Hemacytometer ( )

Impedance Counter ( )

HOW THE GUAVA VIACOUNT ASSAY WORKSThe ViaCount assay uses a proprietary mixture of two

DNA binding dyes to provide sensitive, accurate

detection of viable, apoptotic, and dead cells.

A membrane-permeant dye stains all nucleated

cells, leaving cellular debris unstained so that you can

focus on whole cells. A membrane-impermeant dye

stains only damaged cells (i.e. those with breaches in

the plasma membrane), thus indicating apoptotic and

dying cells.

QUICKLY IDENTIFY APOPTOTIC CELLS With the Guava ViaCount assay, you can easily identify

apoptotic cells using the viability dye (figure on front

and figure 2). You can also use an optional apoptosis

gate in the analysis software that counts apoptotic

cells separately. This optional gate feature enables you

to use the assay for cytotoxicity, apoptosis induction,

or proliferation studies. In these cases, more than a

mere cell number is essential to understanding an

experiment’s outcome.

By contrast, published studies show that trypan

blue exclusion assays often overestimate cell counts2,3.

This may occur because trypan blue cannot identify

apoptotic cells, and so they are added to the count of

viable cells.

Figure 2. The blue population of cells show a significant amount of

annexin V staining (bottom plot), indicating that intermediate levels of

staining with the viability dye correlates with apoptosis.

Nuc

leat

ed C

ells

Viability Stain

Live

Dead

Apoptotic Via

bilit

y Sta

in

Annexin V

Dead

Live

Apoptotic

IMPROVE YOUR CELL-BASED ASSAYSAccurate cell counts and viability measurements make

other functional cellular assays more reproducible

because they provide a more realistic indication of how

many healthy cells are present. For example, ELISPOT

assays show a marked reduction in CVs when cell

concentrations are measured with the Guava ViaCount

assay compared to those done with trypan blue

hemocytometer measurements (Figure 3).

COMPATIBLE WITH MANY CELL TYPESThe Guava ViaCount assay accurately determines

viability and counts for a large variety of cell types.

Tested on a broad selection of mammalian cell lines

(including human, mouse, rat, hamster, and monkey). As

a plus, the assay also works with both primary cells and

whole blood.

3

EASY-TO-USE SOFTWARE Guava’s ViaCount Software Module makes data

collection and analysis even easier. It runs the entire

assay without any need for user input, eliminating

subjectivity. Sample analysis is completed and the data

are automatically exported to a time-stamped

spreadsheet in seconds. Plus, Guava’s new EasyFit

Software is included for computer-aided population

analysis.

The module displays data in user-friendly formats

so you can monitor your experiment or read data right

off the screen. By measuring the staining intensities of

the two dyes, our software represents each cell

counted as a dot on the Cell Dotplot. Cell counts and

percentages are calculated and displayed real-time on

the screen. The optional apoptosis gate (purple line)

can easily be unchecked if only the viable population is

relevant to your research.

Benefits:• Easier to learn and use • Get standardized results even with multiple users • Spend less time optimizing instrument settings • Improve your productivity

ADDITIONAL REAGENTS FOR GREATER FLEXIBILITYCreated to address more difficult cell counting

samples, Guava ViaCount Flex reagent is a more

concentrated formulation of the ViaCount reagent. It

works well for low density cell cultures since it can be

added in small volumes to avoid further sample dilution.

This feature also makes ViaCount Flex reagent ideal for

cell counting and viability assessment in 96-well plates.

For clumped samples, use the ViacCount Cell

Dispersal Reagent, which uses enzymes to gently

disaggretate cells. This helps to improve the accuracy

and precision of cell counts.

ViaCount Flex Reagent• Low density cell samples that are too diluted to be

counted with standard hemacytometers• Cell lines that stain heterogeneously (facilitates gate

settings by providing tighter staining patterns)

Viacount Flex Reagent for Challenging Samples• Non-mammalian cell lines (such as SF9 insect cells)

and other cell lines with unusual culture conditions

Guava ViaCount Cell Dispersal Reagent (CDR) • To reduce clumping in adherent cells

A. Easy viewing of

real-time statistics

for cell counts and

concentrations of

original sample.

B. Optional apoptosis

gate (purple line)

identifies apoptotic

cells (intermediate level

staining, red circle).

C. Real-time counts of

cells in all populations

(apoptotic, dead, viable)

with calculated cell

concentrations.

A

2B 1BC

ORDERING INFORMATIONDescription Catalogue No.

Kits & ReagentsGuava ViaCount Reagent Kit (100 tests) 4000-0040

Guava ViaCount Reagent Kit (600 tests) 4000-0041

Guava ViaCount Flex Reagent for Challenging Samples (100 tests) 4500-0110

Guava ViaCount Cell Dispersal Reagent (100 tests) 4700-0050

Guava ViaCount Flex Reagent (500 tests) 4700-0060

SoftwareViaCount Software, PCA System 0500-1030

ViaCount Software, PCA-96 System 0500-0580

ViaCount Software, EasyCyte MiniSystem 0500-1470

ViaCount Software, EasyCyte Plus System 0500-2000

www.millipore.com

Millipore is a registered trademark of Millipore Corporation. The M mark is a trademark of Millipore Corporation. Guava, the Guava logo, Guava ViaCount, and Guava Technologies are registered trademarks of Guava Technologies, Inc. EasyCyte is a trademark of Guava Technologies, Inc.Lit. No. DS0125EN00 07/08 Printed in U.S.A. BS-GEN-08-00631 © 2008 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

Audience

GuavaAssays & Kits

Flow Cytometry Systems Service

Europe & North America

Academic & Government n n n

Pharma & Biotech l l n

All Other Countries n n n

n Millipore Corporation l Guava Technologies

To learn more about our integrated solutions for

flow cytometry, please contact Millipore or Guava

Technologies. The chart (below) shows which company

serves your organization.

To stay up-to-date on our joint product and

service offerings, please visit

www.millipore.com/flowcytometry.

www.guavatechnologies.com

References

1. Data provided courtesy of Amy Hirst, Raven Biotechnologies, Inc.

2. Mascotti, McCullogh and Burger; Transfusion 40:693-696, 2000.

3. Altman, Rander and Rao; Biotechnology Progress 9:671-674, 1993.

4. Ogasawara K, et al. Impairment of NK cell function by NKG2D modulation in NOD mice. Immunity. 18(1): 41-51 (2003).

5. Arase H, et al. Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science. 296(5571):1323-1326 (2002).

6. Cerwenka A, et al. Cutting edge: the minor histocompatibility antigen H60 peptide interacts with both H-2Kb and NKG2D. Journal of Immunology. 168(7):3131-3134 (2002).

7. Ogasawara K, et al. Inducible costimulator costimulates cytotoxic activity and IFN-gamma production in activated murine NK cells. Journal of Immunology. 169(7):3676-3685 (2002).

8. Phi-Wilson J, et al. A technology for the rapid acquisition of cell number and viability. American Biotechnology Laboratory. pp 34-36 (2001).