ToxCount™
Cell Viability Assay(version A)

Catalog No. 18010

Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
Toll free:877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:+32 (0)2 653 0001
Fax:
+32 (0)2 653 0050
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:
+81 3 5261 8733

www.activemotif.com
www.activemotif.com

iTABLE OF CONTENTS

Page

Overview . . . . . . . �

1Introduction . . . . �

2Advantages of IsoCyte laser scanner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Reagent Preparation and Protocols
Reagent Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
ToxCount Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
IsoCyte Bleed-Through Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Standard Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Imaging the Cells and Data Analysis using IsoCyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
References . . . . . . �

8Appendix
Section A. Optimizing Dye Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section B. ToxCount Protocol for 8-well chamber slide . . . . . . . . . . . . . . . . . . . . . . . . 9
Section C. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section D. Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

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Overview
Active Motif’s ToxCount™ Cell Viability Assay provides reagents to perform a simple two-color
fluorescence cell viability assay. The ToxCount assay is based on the simultaneous determinationof live and dead cells with two probes, calcein AM and ethidium homodimer (EthD-1), that recognize parameters of cell viability including intracellular esterase activity and plasma membrane
integrity, respectively.
Calcein AM is a non-fluorescent cell permeable dye that converts to green fluorescent calcein
in live cells after acetoxymethyl ester hydrolysis by intracellular esterases. Ethidium homodimer(EthD-1) is a red fluorescent nuclear and chromosome counterstain. This reagent does not permeate live cells and is commonly used to detect dead cells in a population. Because the dyes usedin ToxCount are non-fluorescent before interacting with cells, background fluorescence levels arevery low. ToxCount data can be acquired using the BlueShift Biotechnologies IsoCyte™ laser scanner or standard fluorescent microscopes.
product

formatcatalog no.

ToxCount™

20 x 96 rxns*

18010* Sufficient components are provided for performing 20 x 96-well assays using the IsoCyte laser scanner. This assay canalso be easily adapted for use in smaller or larger formats such as 384-well plates or 6-well plates.

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1

Introduction
ToxCount™ provides a two wavelength cell viability assay based on determination of live and deadcells. The assay uses fluorescent dyes that measure permeability of the plasma membrane of dying
cells and the ability of living cells to retain intracellular esterases, respectively. Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymaticconversion of the non fluorescent cell permeable calcein AM to the intensely fluorescent calcein. In
contrast, ethidium homodimer (EthD-1) enters cells with damaged membranes and binds to nucleic
acids where it emits a bright red fluorescent signal in dead cells.
ToxCount can be used on multiple fluorescence detection platforms, including multi-well platescanning, fluorescence microscopy or flow cytometry1. The assay is also suitable for use withmany adherent cell lines2, primary cell types3,4 and small organisms5. This method is less expensive, safer and faster than traditional cell viability assays such as 51Cr-release, LDH release andtrypan blue exclusion. The calcein AM and EthD-1 fluorescent dyes have been used to study
apoptosis2 and the cytotoxic effects of numerous agents including viral proteins6, oxalate7, amyloid proteins8 and cells such as lymphocytes1. The assay is ideally suited for rapid screening oflibraries of drugs and small molecules for cytotoxic effects and is particularly applicable to highcontent screening using the BlueShift IsoCyte laser scanner.
The dyes provided in ToxCount are easy to use, requiring only 30 minutes loading time. In addition,these dyes can be used in homogeneous ‘no-wash’ protocols as they are non-fluorescent in solution.
Active Motif’s ToxCount Kit has been optimized specifically for scanning on the IsoCyte laser scanner. A
detailed protocol is available for customers who wish to use the reagents for 96-well, high throughput
cytotoxicity assays with the IsoCyte and automated analysis software will rapidly count the number oflive and dead cells, providing the user with an easily interpreted readout for each well.

Advantages of the IsoCyte™ laser scanner
The IsoCyte laser scanner from BlueShift Biotechnologies is a bench top laser scanning fluorimeterthat measures fluorescence emission signals and their characteristic properties other than intensity and wavelength (i.e. fluorescence polarization). The platform has been designed to provide
unprecedented flexibility in assay applications, sample formats and fluorophore compatibility. The
system scans microplates with industry standard Society of Biomolecular Screening (SBS) allowing
measurements to be made from 96, 384 or 1536 well plates.
The scanner’s object based technology measures signals associated with objects in the entire wellenabling the researcher to:• Distinguish object signal from background• Measure object fluorescence intensity• Provide information regarding cell number, area and location in the wellwww.activemotif.com

2

Kit Components and Storage
Kit reagents are supplied lyophilized and are stable at room temperature. Once the EthD-1 isresuspended in DMSO it must be stored at -20°C. Once the calcein AM has been resuspended inanhydrous DMSO it must be aliquoted, desiccated, protected from light and stored at -20°C as
indicated in the Buffer Preparation Section on page 4.
Reagents

Quantity

Storage / Stability

calcein AM

110 µl (1 mM)(lyophilized)

RT for 6 months

EthD-1 (Ethidium homodimer)

110 µl (1 mM)(lyophilized)

RT for 6 months

Additional materials required• DMSO (anhydrous)• Serum-free media• 37°C incubator• Saponin (Sigma, Cat. No. S4521-25G)• PBS with calcium and magnesium supplemented with 10 mM glucose• Light microscope• Fluorescence microscope/reader or IsoCyte laser based scanner from Blueshift
Biotechnologies (Sunnyvale, CA)• Suitable tissue culture plates such as 96 well, clear bottom, black walled sterile polystyrene
plates (i.e. CoStar catalog # 3904)

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Reagent Preparation and Protocols
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Preparation of calcein AM Stock Solution
The calcein AM reagent is supplied lyophilized. Prepare a 1 mM calcein AM stock solution byadding 110 µl of anhydrous DMSO to the lyophilized calcein AM in the provided vial. Aliquot in
5 µl fraction and store at -20˚C desiccated. On the day of the experiment, you will prepare anaqueous working solution containing calcein AM immediately prior to use, and use within one
day. Note: Reagent must be desiccated once it is resuspended in DMSO as it is susceptible tohydrolysis when exposed to moisture.
Preparation of EthD-1 Stock Solution
The EthD-1 is supplied lyophilized. Prepare a 1 mM EthD-1 stock solution by adding 110 µl of DMSO
(does not have to be anhydrous) to the lyophilized EthD-1 in the provided vial. Store at -20˚C.
EthD-1 is stable and insensitive to moisture. Stock solutions of EthD-1 can be stored frozen at-20˚C for at least one year.
Selection of Optimal Filters
Calcein AM has an excitation/emission spectrum of 496 nm and 516 nm, respectively. A 510-540
nm band pass filter can be used for calcein AM emission. EthD-1 excites at 528 nm and emits at
598 nm. A conventional 600 nm long pass filter can be used to image this dye and to block bleed
through from the green channel. If using the IsoCyte, it has a 560 nm dichroic filter to allow
transmission of wavelengths longer than 560 nm to the red detection channels.
Preparation of the Cells
Adherent cells may be cultured directly on the microtitre plate. For a 96-well plate used with
IsoCyte, we recommend seeding cells at 2.5 x 103 cells per well in 200 µl of supplemented
medium. Incubate cells at 37˚C until 60% confluent. NOTE: After adding the cells to the plateleave the plate undisturbed in the hood for 20 minutes before placing it in the incubator. Moving
the plate immediately after seeding can generate forces that push cells to the outer edges of thewell. If using suspension cells, please see Appendix Section C. Troubleshooting on page 11.
Preparation of Saponin Solution
Prepare a 0.1% solution of saponin by dissolving 0.05 g of saponin in 50 ml PBS containing calcium
and magnesium or serum free medium. This solution is stable for at least 48 hours.
Preparation of PBS with 10 mM glucose (PBSg)
Prepare PBSg by dissolving 0.09 g of glucose in 50 ml PBS containing calcium and magnesium. We
recommend preparing fresh PBSg each day an experiment is to be performed.

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Typical ToxCount Experimental Design
The ToxCount protocol volumes are based on a 96-well plate. Sufficient reagents are provided
for performing 20 x 96-well plates. However, the assay is also suitable for use with other formats
as indicated in Table 1 below.
Plate type

Total vol/well(including
test drug/
saponin) (µl)

Vol. dye/well(µl)

Vol. medium/
plate (ml)calcein AM
required/plate (µl)

EthD-1required/plate (µl)# platespossible

1536

5

2.5

4

4

4

25

384

50

25

10

10

10

10

96

100

50

5

5

5

20

24

500

250

6

6

6

16

12

1000

500

6

6

6

16

6

2000

1000

6

6

6

16

Table 1. A schematic of volumes required in the ToxCount Assay. The protocol is based on a 96-well plate with IsoCyte.

Table 2 below shows a typical experiment which includes individual staining controls for bothdyes in treated and untreated wells (to get a readout for background in each channel), and wells
that were treated or untreated and stained with both dyes. Once this experiment has been performed for a certain cell type and the background values are known, further experiments can be
performed using the Standard Protocol on page 6.
1

2

3

4

5

6

7

8

9

10

Calcein
AM Livecells

Calcein
AM
Dead
cells
10 minSaponinCalceinAM
Deadcells 10
min Test
Reagent

EthD-1Live
cells

EthD-1Deadcells
10 minSaponin

EthD-1Deadcells 10
min Test
Reagent

EthD-1 +Calcein
AM Livecells

EthD-1 +Calcein
AM
Dead
cells
10 minSaponin

EthD-1 +CalceinAM
Deadcells 10
min Test
Reagent

No Dyes

Table 2. A typical cytotoxicity/cell viability experiment using ToxCount assay reagents. Cells that have been treatedwith saponin should not be stained with calcein AM and untreated cells should not stain with EthD-1. The numbers indicate separate columns of a 96 well plate.

IsoCyte Bleed-Through Test
When using the IsoCyte, it is necessary to assess the extent of bleed-through from one channel to another or background levels (according to the Table 2 above). For this test, only 2 ml ofcalcein AM solution or EthD-1 solution or a 2 ml solution with a mixture of both dyes is prepared.
Once this bleed-through experiment has been performed for a certain cell type and the background values are known, further experiments can be performed using the standard protocol on
page 6.

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NOTE:All working solutions can be prepared with either PBSg or serum free medium. For
the IsoCyte, no background interference has been seen from phenol red in the media,
however, there may be interference when using a fluorescent microscope.

Step 1: Treatment of cells to induce cell death.
1.

Prepare a 0.1% saponin solution by dissolving 0.05 g saponin in 50 ml PBSg or serum-free
medium.

2.

Remove the media from the wells of the microplate to be tested using a multichannel
pipettor.

3.

Add 50 µl of the test compounds or saponin solution to the wells designated as “dead”
cells for the desired amount of time. For 0.1% saponin we recommend a 10 minute 37°C
treatment.

4.

Add 50 µl of PBSg or serum free medium to the “live” cells for the desired amount of time.
NOTE:

The calcein AM and EthD-1 are supplied lyophilized and instructions for reconstituting
are on page 4. If you have already reconstituted the dyes, please ensure the calcein AM
and EthD-1 dyes have been thawed for at least 5 minutes and are at room temperature
prior to beginning the Step 2 below. This helps avoid the dilution of the solution byambient moisture condensation. Protect the dyes from light as much as possible.

Step 2: Preparation of 1 µM calcein AM working solution.
1.

The calcein AM is supplied lyophilized. Please read the section on page 4 about reconstituting andpreparing the calcein AM stock solution. Prepare a 1 µM calcein AM working solution by adding 2 µlof the 1 mM calcein AM stock solution to 2 ml of PBSg or serum free medium.

Step 3: Preparation of 1 µM EthD-1 working solution.
1.

The EthD-1 is supplied lyophilized. Please read the section on page 4 about reconstituting
and preparing the EthD-1 stock solution. Prepare a 1 µM EthD-1 working solution by adding 2µl of the 1 mM EthD-1 stock solution to 2 ml of PBSg or serum free medium.

Step 4: Preparation of a mixture of 1 µM calcein AM and 1 µM EthD-1.
1.

The calcein AM and EthD-1 are supplied lyophilized. Please read the section on page 4 aboutreconstituting and preparing the stock solutions. Prepare a mixture of 1 µM calcein AM and1 µM EthD-1 by adding 2 µl of 1 mM calcein AM stock solution and 2 µl of 1 mM EthD-1 prepared in the previous section to 2 ml PBSg or serum free medium.

Step 5: Perform the Viability Assay.
1.

Treat cells with the test compounds or saponin for the desired amount of time.

2.

Add 50 µl of 1 µM calcein AM to wells designated “calcein AM only.”3.

Add 50 µl of 1 µM EthD-1 to wells designated “EthD-1 only.”4.

Add 50 µl of the of 1 µM calcein AM/1 µM EthD-1 mixture to wells designated “EthD-1 + calcein AM”

5.

Incubate cells at 37˚C for 30 minutes.

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Standard Protocol
If using the IsoCyte, proceed with the following Standard Protocol after completion of theBleed-Through Test for a specific cell type. These volumes are based on a 96-well plate. If using a
different size plate, please refer to Table 1 on page 5.
NOTE:

All working solutions can be prepared with either PBSg or serum free medium. For
the IsoCyte, no background interference has been seen from phenol red in the media,
however, there may be interference when using a fluorescent microscope.

Step 1: Treatment of cells to induce cell death1.

Prepare a 0.1% saponin solution by dissolving 0.05 g saponin in 50 ml PBSg or serum-free
medium.

2.

Remove the media from the wells of the microplate to be tested using a multichannel
pipettor.

3.

Add 50 µl of the test compounds or saponin solution for the desired amount of time. For
0.1% saponin we recommend a 10 minute 37°C treatment.
NOTE:

The calcein AM and EthD-1 are supplied lyophilized and instructions for reconstituting are on page 4. If you have already reconstituted the dyes, please ensure
the calcein AM and EthD-1 dyes have been thawed for at least 5 minutes and areat room temperature prior to beginning the Step 2 below. This helps avoid the
dilution of the solution by ambient moisture condensation. Protect the dyes
from light as much as possible.

Step 2: Perform the Viability Assay
1.

The calcein AM and EthD-1 are supplied lyophilized. Please read the section on page 4 aboutreconstituting and preparing the stock solutions. Prepare a mixture of 1 µM calcein AM and1 µM EthD-1 by adding 5 µl of 1 mM calcein AM stock solution and 5 µl of 1 mM EthD-1 stock
solution to 5 ml PBSg or serum free medium.

2.

Add 50 µl of dye mixture to each well.

3.

Incubate cells at 37˚C for 30 minutes.

4.

Proceed to cell imaging.

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7

Imaging the cells and Data Analysis using the IsoCyte
The staining of the cells can be checked on a fluorescent microscope before scanning on the
IsoCyte. There is no need to remove the dye solution from the well or wash the cells since thedyes are not fluorescent unless they are inside the cell.
Instructions for scanning plates on the IsoCyte
1

Turn on the scanner using the On/Off (I/O) switch located on the left side of the instrument. Wait 5 minutes for the laser to warm up.

2.

Start the BBIsoCyte software by double-clicking on the icon on the desktop.

3.

Click on the blue arrow shaped icon on the tool bar to eject the plate holder.

4.

Insert plate and click icon again to close.

5.

Open “ToxCount” method.

6.

Select the wells that are to be scanned. The wells will be highlighted in blue if they havebeen selected.

7.

Click on the scan icon.

8.

When the scan has finished save the data file.

9.

When the analysis is complete, a new folder containing the .csv files will appear in thesame directory as the bbx files.

References
1.
2.
3.
4.
5.
6.
7.
8.

Papadopoulos N.G., et al. (1994) Immunol Methods 177(1-2):101-11.
Lucas R. et al. (1998) Blood 92(12):4730-41.
Decherchi P., et al. (1997) Neurosci Methods 71: 205-213.
Weston S.A. and Parish C.R. (1992) Cytometry 13: 739-749.
Kaneshiro E.S., et al. (1993) J Microbiol Methods 17: 1
Guzman E. and McCrae M.A. (2005) J Virol Methods 127(2):119-25.
Kohjimoto Y. (1999) Kidney Int. 56(4):1432-41.
Liu M.L. and Hong S.T. (2005) Exp Mol Med. 37(6):559-66.

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Appendix
Section A. Optimizing Dye Concentrations
Determination of Optimal Dye Concentrations
Using the IsoCyte, we have determined that 0.5 µM of calcein AM and EthD-1 provide optimal
results for staining live or dead cells. However, depending on the cell type assayed or if you areusing a fluorescent microscope for detection, optimal dye concentrations may vary. The following method can be used to determine the optimal dye concentration.
1.

Remove the ToxCount assay reagents from the freezer and allow them to warm to room
temperature.

2.

Prepare samples of live cells as well as dead cells on a 96-well plate. Induce cell death using
0.1% saponin for 10 minutes at 37°C.

3.

Using samples of dead cells, select an EthD-1 concentration that stains the dead cell nuclei
bright red without staining the cytoplasm significantly (recommend 0.1 to 10 µM EthD-1).

4.

Using samples of dead cells, select a calcein AM concentration that does not give significant fluorescence in the dead cell cytoplasm (recommend 0.1 to 10 µM calcein AM).

5.

Using samples of live cells, check to see that the calcein AM concentration selected above
generates sufficient fluorescence in the live cells.

6.

The reagent concentrations determined in Step 3 and 5 are optimal for the viability experiments.

Section B. ToxCount Protocol for 8-well chamber slide
If you are using an 8-well chamber slide and evaluating cell viability through use of a fluorescent
microscope, please follow the protocol listed below.
1.

Seed cells in 250 µl of culture medium at a density of 1 x 104 cells per well in an 8 wellchamber slide.

2.

Incubate cells overnight at 37°C, 5% CO2.

3.

Treat cells with 125 µl 0.1% saponin for 10 minutes or with test compounds for desired
amount of time.

4.

Prepare a 2X solution of calcein AM and EthD-1 by adding 1 µl from each of the 1 mM stocks
to 1 ml of serum free media or PBSg.

5.

Add 125 µl of the dye mixture to each well and leave for 30 minutes at 37°C.

6.

Image slide on microscope.

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9Section C. Troubleshooting Guide
PROBLEM/QUESTIONRECOMMENDATIONNo staining in sample wells

Use a microscope with a bright field light to check that the cells are still present
on the plate and have not lifted off the surface as a result of treatment with
toxic reagents.
Increase the exposure time on the microscope or the gain setting on the
IsoCyte.
Label cells with a higher dye concentration

High background (cell death Increase concentration of saponin or try an alternative toxic agent such as
not induced)
methanol to induce cell deathCan I use suspension cells
rather than adherent cells?

Yes. Wash suspension cells with 500 -1000 volumes of PBS and sediment by centrifugation. Plate cells in desired plate size in serum free medium to 60% confluency. After cells have settled, treat with 2X solution of test reagent or saponin.
Prepare a 3X Dye solution (1.5 µM) and add to wells (final concentration will be
0.5 µM). Incubate for 30 minutes and analyze as desired.

Section D. Related ProductsProtein Transfection

Unit

Catalog No.

Chariot™

25 rxns
100 rxns

30025
30100

Fluorescent Dyes

Unit

Catalog No.

Chromeo™ 494 Carboxylic Acid

1 mg
5 mg
1 mg
5 mg
1 mg
5 mg
1 mg
5 mg
1 mg
5 mg
1 mg
5 mg
1 mg
5 mg
1 mg
5 mg
1 mg
5 mg
1 mg
5 mg
1 mg
5 mg
1 mg
5 mg

15110
16110
15111
16111
15112
16112
15113
16113
15210
16210
15211
16211
15212
16212
15213
16213
15310
16310
15311
16311
15312
16312
15313
16313

1 kit
1 kit
1 kit
1 kit
300 rxns
300 rxns

34001
34004
34005
34006
34101
34104

Chromeo™ 494 NHS-Ester
Chromeo™ 494 Biotin
Chromeo™ 494 Streptavidin
Chromeo™ 546 Carboxylic Acid
Chromeo™ 546 NHS-Ester
Chromeo™ 546 Biotin
Chromeo™ 546 Streptavidin
Chromeo™ 642 Carboxylic Acid
Chromeo™ 642 NHS-Ester
Chromeo™ 642 Biotin
Chromeo™ 642 Streptavidin
Protein Labeling
LigandLink™ pLL-1 Kit
LigandLink™ pLL-1-NFkB p65 Kit
LigandLink™ pLL-1-p53 Kit
LigandLink™ pLL-1-STAT1 Kit
LigandLink™ Fluorescein Label
LigandLink™ Hexachlorofluorescein Label

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10Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:+32 (0)2 653 0001
Fax:
+32 (0)2 653 0050
E-mail:eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
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