ELEVATE DRUG DISCOVERY

Advance Detection of Cytotoxicity A wide range of solutions for predicting cytotoxicity earlier in your drug discovery process

Development of predictive in vitro assays for early toxicity evaluation streamlines the drug development process and

reduces costly drug attrition rates during clinical development. Assays that use induced pluripotent stem cells (iPSCs)

provide more biologically relevant cell-based models and are emerging as efficient tools for evaluating safety and

toxicity earlier in the drug development process. iPSC-derived cardiomyocytes, hepatocytes, and neurons have typical

phenotypic characteristics and function in similar fashion to the primary cells they represent.

Cardiomyocyte beating and cytotoxicity assays . . . . . . . . . 2

Monitoring BNP expression and cell size to determine

hypertrophic response in human cardiomyocytes . . . . . . . . 3

Biologically relevant cardiotoxicity

data early in the drug discovery process . . . . . . . . . . . . . . . 4

Characterization of hERG channel blockers . . . . . . . . . . . . . 5

Measuring cell viability and cytotoxicity . . . . . . . . . . . . . . . . .6

Toxicity assays using iPSC-derived hepatocytes . . . . . . . . . 7

Characterizing the viability and morphology

of human iPSC-derived neuronal cultures . . . . . . . . . . . . . . . 8

Visualizing subcellular vesicles to

quantitate autophagy in neuronal cells . . . . . . . . . . . . . . . . .9

Case Study: Assessment of cardiotoxicity

hazard of environmental compounds . . . . . . . . . . . . . . . . . 10

Case Study: Effects of petroleum substances on

iPSC-derived cardiomyocytes and hepatocytes. . . . . . . . . 11

2 moleculardevices.com/celltox

Stem cell-derived human cardiomyocytes have phenotypic characteristics and

electrophysiological profiles similar to those of native human cardiac cells.

Cardiomyocytes in culture are able to form a beating syncytium, which behaves

similarly to native cardiomyocytes. Oscillation of intracellular calcium levels occurring

with synchronized contractions of the cells can be monitored using a calcium-

sensitive dye, and treatment-induced changes in the pattern of oscillation can be

monitored via changes in fluorescent signal over time.

Recently, it has been shown that changes in intracellular calcium flux associated

with cardiomyocyte contractions can be monitored on fluorescence imaging plate

readers. The SpectraMax i3x Multi-Mode Microplate Platform with SpectraMax

MiniMax 300 Imaging Cytometer enables detection of cardiomyocyte beating and

fluorescence imaging of cell viability in a single instrument. Compound-induced

changes in observed beating rate can be correlated to cell viability for a more

accurate assessment of cardiotoxicity.

Comparing peak data to cell viability. Several compounds that dramatically reduced the peak count were shown to have little or no effect on overall cell viability. For example, digoxin, a drug widely used in the treatment of arrhythmias, reduced peak count from the roughly 12 beats per 25-second reading down to 1 or 2 beats. However, cell covered area was 71% compared to 82% in DMSO-treated controlsnot a significant reduction in cell viability. Based on results from the library screen, compounds with observed effects on beating rate and cytotoxicity were selected for more detailed study.

Captured on the SpectraMax i3 with MiniMax 300 Imaging Cytometer

Captured on the ImageXpress Micro XLS Widefield High-Content Analysis System

Click image to play video

Captured on the ImageXpress Micro XLS Widefield High-Content Analysis System

Click image to play video

Captured on the SpectraMax i3 with MiniMax 300 Imaging Cytometer

Benefits Obtain more biologically

relevant data by combining microplate reader assays with imaging cytometry

No imaging expertise required with intuitive SoftMax Pro Software user interface

Monitor iPSC cardiomyocyte cell contraction with the use of a calcium-sensitive dye

Cardiomyocytes

Cardiomyocyte beating and cytotoxicity assays

Figure 2. Comparing peak data to cell viabilitySeveral compounds that dramatically reduced the peak count were shown to have little or no effect on overall cell viability. For example, digoxin, a drug widely used in the treatment of arrhythmias, reduced peak count from the roughly 12 beats per 25-second reading down to 1 or 2 beats. However, cell covered area was 71% compared to 82% in DMSO-treated controlsnot a significant reduction in cell viability. The dopamine inverse agonist haloperidol had a distinct effect on beating profile without impacting viability. Other compounds, like staurosporine, greatly reduced both cell viability and peak count (Figure 2).

Based on results from the compound library screen, compounds with observed effects on beating rate and cytotoxicity were selected for more detailed study. Representative compounds were added to cardiomyocytes in a 1:3 dilution series to determine IC50 values. As before, cardiomyocyte contractions were visualized using the EarlyTox Cardiotoxicity dye, and beating profiles measured on the SpectraMax i3 Platform.

Detailed views of kinetic traces and images of cells treated with DMSO (control), digoxin, haloperidol, or staurosporine. Digoxin and haloperidol decreased the number of peaks measured in beating cells but had no significant effect on viability, while staurosporine negatively affected both beating and viability.

Figure 2. Comparing peak data to cell viabilitySeveral compounds that dramatically reduced the peak count were shown to have little or no effect on overall cell viability. For example, digoxin, a drug widely used in the treatment of arrhythmias, reduced peak count from the roughly 12 beats per 25-second reading down to 1 or 2 beats. However, cell covered area was 71% compared to 82% in DMSO-treated controlsnot a significant reduction in cell viability. The dopamine inverse agonist haloperidol had a distinct effect on beating profile without impacting viability. Other compounds, like staurosporine, greatly reduced both cell viability and peak count (Figure 2).

Based on results from the compound library screen, compounds with observed effects on beating rate and cytotoxicity were selected for more detailed study. Representative compounds were added to cardiomyocytes in a 1:3 dilution series to determine IC50 values. As before, cardiomyocyte contractions were visualized using the EarlyTox Cardiotoxicity dye, and beating profiles measured on the SpectraMax i3 Platform.

Detailed views of kinetic traces and images of cells treated with DMSO (control), digoxin, haloperidol, or staurosporine. Digoxin and haloperidol decreased the number of peaks measured in beating cells but had no significant effect on viability, while staurosporine negatively affected both beating and viability.

Figure 2. Comparing peak data to cell viabilitySeveral compounds that dramatically reduced the peak count were shown to have little or no effect on overall cell viability. For example, digoxin, a drug widely used in the treatment of arrhythmias, reduced peak count from the roughly 12 beats per 25-second reading down to 1 or 2 beats. However, cell covered area was 71% compared to 82% in DMSO-treated controlsnot a significant reduction in cell viability. The dopamine inverse agonist haloperidol had a distinct effect on beating profile without impacting viability. Other compounds, like staurosporine, greatly reduced both cell viability and peak count (Figure 2).

Based on results from the compound library screen, compounds with observed effects on beating rate and cytotoxicity were selected for more detailed study. Representative compounds were added to cardiomyocytes in a 1:3 dilution series to determine IC50 values. As before, cardiomyocyte contractions were visualized using the EarlyTox Cardiotoxicity dye, and beating profiles measured on the SpectraMax i3 Platform.

Detailed views of kinetic traces and images of cells treated with DMSO (control), digoxin, haloperidol, or staurosporine. Digoxin and haloperidol decreased the number of peaks measured in beating cells but had no significant effect on viability, while staurosporine negatively affected both beating and viability.

Figure 2. Comparing peak data to cell viabilitySeveral compounds that dramatically reduced the peak count were shown to have little or no effect on overall cell viability. For example, digoxin, a drug widely used in the treatment of arrhythmias, reduced peak count from the roughly 12 beats per 25-second reading down to 1 or 2 beats. However, cell covered area was 71% compared to 82% in DMSO-treated controlsnot a significant reduction in cell viability. The dopamine inverse agonist haloperidol had a distinct effect on beating profile without impacting viability. Other compounds, like staurosporine, greatly reduced both cell viability and peak count (Figure 2).

Based on results from the compound library screen, compounds with observed effects on beating rate and cytotoxicity were selected for more detailed study. Representative compounds were added to cardiomyocytes in a 1:3 dilution series to determine IC50 values. As before, cardiomyocyte contractions were visualized using the EarlyTox Cardiotoxicity dye, and beating profiles measured on the SpectraMax i3 Platform.

Detailed views of kinetic traces and images of cells treated with DMSO (control), digoxin, haloperidol, or staurosporine. Digoxin and haloperidol decreased the number of peaks measured in beating cells but had no significant effect on viability, while staurosporine negatively affected both beating and viability.

Resources

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Cardiomyocyte beating and cytotoxicity assays on the SpectraMax i3 Multi-Mode Microplate Platform with SpectraMax MiniMax Imaging Cytometer

Download Application Note:

High throughput cardiotoxicity assays using stem cell-derived cardiomyocytes

Review Article Abstract:

Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity

3moleculardevices.com/celltox

Cardiac hypertrophy is a condition associated with many heart diseases such as

myocardial infarction, ischemia, hypertension, valvular dysfunction, and is also

observed as a toxic side effect of environmental contaminants or pharmaceutical drug

candidates. It is characterized by different cellular changes including increased cell

size and enhanced protein synthesis. One of the classic biomarkers for hypertrophy is

B-type natriuretic peptide (BNP), which is over produced in hypertrophic cardiac cells.

Monitoring induced pluripotent stem cell iPSC-derived cardiomyocytes with high-

content imaging assays enables high-throughput measurement of the onset or

amelioration of hypertrophy as a result of compound toxicity or as a screening

endpoint, respectively. Thus, these assays are a robust and useful tool for phenotypic

screening processes used in both pharmacological testing and for drug development

through disease modeling.

Resources

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Monitoring BNP Expression and Cell Size to Determine Hypertrophic Response in Human Cardiomyocytes

Benefits Visualize changes in

morphology

Quantify multiple hypertrophic parameters at once

Maximize throughput by screening cardiomyocytes in 96- and 384-well plates

Collect statistically significant sample sizes using adaptive acquisition

Monitoring BNP expression and cell size to determine hypertrophic response in human cardiomyocytes

Figure 2A. BNP expression in iCell Cardiomyocytes before and after ET-1 addition. Images of iCell Cardiomyocytes untreated (left) or stimulated with 10 nM ET-1 for 18 hours (right). Cells were labeled with antibodies to detect BNP expression (red), and nuclei were stained with Hoechst 33342 (blue). Images acquired at 20x magnification. The area of BNP signal was used to calculate the levels of BNP expression1.

50 m 50 m

Figure 2B. In this representative 384-well experiment, a dose-dependent increase in BNP expression occurred after stimulation with the indicated concentrations of ET-1. The EC50 value for ET-1 was 24 pM (mean SEM; n = 4 for each point on the curve)1.

Figure 3. Images of untreated (left) and 10 nM ET-1induced (middle) iCell Cardiomyocytes illustrate differences in cell size. Alexa Fluor 488labeled phalloidin was used to determine cell body size (green), and nuclei were stained with Hoechst 33342 (blue). Images were acquired at 20x magnification. Changes in cell size (square microns) were quantified against a titration of ET-1, with an EC50 value of 11 pM (mean +/- SEM; n=3) (right)2.

100 m 100 m

BNP expression in ET-1induced cardiomyocytes

Log [ET-1] (M)

BNP

Expr

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Cell size change in hypertrophic cardiomyocytes

Tota

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Log [ET-1] (M)

Quantifying Changes in Molecular BiomarkersIncreased levels of BNP is one of many proteins whose expression is altered during cardiac hypertrophy. Here, the expression of BNP was determined using high-content imaging by labeling BNP with a fluorescent antibody to measure cytoplasmic BNP staining (Figure 2A). In this experiment, iCell Cardiomyocytes were treated with a 10-point dilution series of ET-1. A dose response curve was generated for the ET-1 induced expression of BNP in cardiomyocytes by plotting the BNP signal per cell against the log concentration of compound treatment (Figure 2B). The curve was then fit to generate an EC50 value from the ET-1 dose-response.

Quantifying Changes in Cell Size Increased cell size and structural reorganization of the actin cytoskeleton are well-accepted cellular features of the hypertrophic response2. To illustrate how hypertrophy can be determined by monitoring increases in cell size, iCell Cardiomyocytes were induced with ET-1, stained and then imaged on the ImageXpress Micro System. Hypertrophic cardiomyocytes were visibly larger than untreated cells. Increases in cell size were quantified using the MetaXpress Software (Figure 3) and resulted in a similar EC50 value to that for BNP detection.

Bringing High-Throughput Screening and Human Biology to Early Drug DiscoveryHypertrophy and heart failure are common therapeutic areas of drug discovery. The high throughput capabilities of the ImageXpress Micro XLS System combined with human stem cell-derived tissue cells provide a complete system for accessing human biology early in the drug discovery screening process. As a proof

Figure 2A. BNP expression in iCell Cardiomyocytes before and after ET-1 addition. Images of iCell Cardiomyocytes untreated (left) or stimulated with 10 nM ET-1 for 18 hours (right). Cells were labeled with antibodies to detect BNP expression (red), and nuclei were stained with Hoechst 33342 (blue). Images acquired at 20x magnification. The area of BNP signal was used to calculate the levels of BNP expression1.

50 m 50 m

Figure 2B. In this representative 384-well experiment, a dose-dependent increase in BNP expression occurred after stimulation with the indicated concentrations of ET-1. The EC50 value for ET-1 was 24 pM (mean SEM; n = 4 for each point on the curve)1.

Figure 3. Images of untreated (left) and 10 nM ET-1induced (middle) iCell Cardiomyocytes illustrate differences in cell size. Alexa Fluor 488labeled phalloidin was used to determine cell body size (green), and nuclei were stained with Hoechst 33342 (blue). Images were acquired at 20x magnification. Changes in cell size (square microns) were quantified against a titration of ET-1, with an EC50 value of 11 pM (mean +/- SEM; n=3) (right)2.

100 m 100 m

BNP expression in ET-1induced cardiomyocytes

Log [ET-1] (M)

BNP

Expr

essi

on

Cell size change in hypertrophic cardiomyocytes

Tota

l Cel

l Are

a

Log [ET-1] (M)

Quantifying Changes in Molecular BiomarkersIncreased levels of BNP is one of many proteins whose expression is altered during cardiac hypertrophy. Here, the expression of BNP was determined using high-content imaging by labeling BNP with a fluorescent antibody to measure cytoplasmic BNP staining (Figure 2A). In this experiment, iCell Cardiomyocytes were treated with a 10-point dilution series of ET-1. A dose response curve was generated for the ET-1 induced expression of BNP in cardiomyocytes by plotting the BNP signal per cell against the log concentration of compound treatment (Figure 2B). The curve was then fit to generate an EC50 value from the ET-1 dose-response.

Quantifying Changes in Cell Size Increased cell size and structural reorganization of the actin cytoskeleton are well-accepted cellular features of the hypertrophic response2. To illustrate how hypertrophy can be determined by monitoring increases in cell size, iCell Cardiomyocytes were induced with ET-1, stained and then imaged on the ImageXpress Micro System. Hypertrophic cardiomyocytes were visibly larger than untreated cells. Increases in cell size were quantified using the MetaXpress Software (Figure 3) and resulted in a similar EC50 value to that for BNP detection.

Bringing High-Throughput Screening and Human Biology to Early Drug DiscoveryHypertrophy and heart failure are common therapeutic areas of drug discovery. The high throughput capabilities of the ImageXpress Micro XLS System combined with human stem cell-derived tissue cells provide a complete system for accessing human biology early in the drug discovery screening process. As a proof

Figure 2A. BNP expression in iCell Cardiomyocytes before and after ET-1 addition. Images of iCell Cardiomyocytes untreated (left) or stimulated with 10 nM ET-1 for 18 hours (right). Cells were labeled with antibodies to detect BNP expression (red), and nuclei were stained with Hoechst 33342 (blue). Images acquired at 20x

of BNP expression1 .

50 m 50 m

Figure 2B. In this representative 384-well experiment, a dose-dependent increase in BNP expression occurred after stimulation with the indicated concentrations of ET-1. The EC

50

value for ET-1 was 24 pM (mean SEM; n = 4 for each point on the curve)1 .

Figure 3. Images of untreated (left) and 10 nM ET-1induced (middle) iCell Cardiomyocytes illustrate differences in cell size. Alexa Fluor 488labeled phalloidin was used to determine cell body size (green), and nuclei were stained with Hoechst 33342 (blue). Images were acquired at 20x

an EC50 value of 11 pM (mean +/- SEM; n=3) (right)2.

100 m 100 m

BNP expression in ET-1induced cardiomyocytes

Log [ET-1] (M)

BNP Exp

ression

Cell size change in hypertrophic cardiomyocytes

Total C

ell Area

Log [ET-1] (M)

A. BNP expression in iCell Cardiomyocytes before and after ET-1 addition. Images of iCell Cardiomyocytes untreated (left) or stimulated with 10 nM ET-1 for 18 hours (right). Cells were labeled with antibodies to detect BNP expression (red), and nuclei were stained with Hoechst 33342 (blue). Images acquired at 20x magnification. The area of BNP signal was used to calculate the levels of BNP expression.

A

B

B. Images of untreated (left) and 10 nM ET-1induced (right) iCell Cardiomyocytes illustrate differences in cell size. Alexa Fluor 488labeled phalloidin was used to determine cell body size (green), and nuclei were stained with Hoechst 33342 (blue). Images were acquired at 20x magnification. Changes in cell size (square microns) were quantified against a titration of ET-1, with an EC

50 value of 11 pM (mean +/- SEM; n=3).

Cardiomyocytes

4 moleculardevices.com/celltox

Assessment of cardiotoxicity is important in the early stages of drug discovery to

enable elimination of potentially toxic compounds from further development. This

evaluation is critical to reduce the inefficiencies and high costs associated with

compounds that fail during cardiac safety assessment. There is a growing need for

highly predictive in vitro cardiotoxicity assays that use biologically relevant cell-based

models and are suitable for high-throughput screening. iPSC-derived cardiomyocytes

are especially attractive cell models because they represent gene expression profiles

as well as phenotypic characteristics similar to native cardiac cells.

The calcium sensitive dye in the EarlyTox Cardiotoxicity Kit makes it possible to

evaluate concentration-dependent modulation of calcium peak frequency and

illustrate oscillation patterns in iPSC-derived cardiomyocytes using either the FLIPR

Tetra System or the SpectraMax i3x Multi-Mode Microplate Reader. Identified

changes in parameters such as peak frequencies, amplitude, peak width, rise and

decay times enable the ability to predict possible toxicity or eliminate compounds

from consideration that might otherwise be put forward to medicinal chemistry or to

preclinical development.

Resources

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EarlyTox Cardiotoxicity Kit provides biologically relevant cardiotoxicity data earlier in the drug discovery process

Download Application Note:

Compound effects upon calcium transients in beating Axiogenesis Cor.4U human iPSC-derived cardiomyocytes

iPSC-derived cardiomyocytes treated with sotalol show decreased beat frequency and altered beat pattern. Sotalol is a non-selective competitive -adrenergic receptor blocker that also inhibits hERG. In addition to slowing peak frequency of calcium in iPSC-derived cardiomyocytes, it alters the normal beat pattern as compared to the untreated control.

Benefits Evaluate compound toxicity

and efficacy earlier in the drug discovery process

Analyze cardiotoxicity profiles in a biorelevant system

Scale assay size to meet throughput requirements

Cardiomyocytes

Biologically relevant cardiotoxicity data early in the drug discovery process

Materials and methodsIn this assay, iCell Cardiomyocytes, human induced pluripotent stem cell-derived (iPSC) cardiomyocytes from Cellular Dynamics International were cultured according to their suggested protocol. Cells were plated in a 384-well black-walled clear plate 1014 days prior to experiments. EarlyTox Cardiotoxicity Explorer Kit (R8210) dye loading buffer was warmed to 37C to avoid slowing the beating of the cardiomyocytes upon addition. 25 L dye was added to 25 L cells in the plate. Each plate was incubated at 5% CO

2, 37C for two hours.

Three reference compounds that are included in the kit were studied to demonstrate a variety of effects upon the iPSC cardiomyocytes: propranolol, isoproterenol and sotalol. A compound plate containing concentration responses was made up at 5X final concentration in Component B buffer and warmed to 37C prior to addition to minimize temperature effects upon the cells. After compound addition on the FLIPR Tetra System, cell beat rates and patterns can be monitored for up to 46 hours. Plates were read again 10 minutes post compound addition. Additional reads were taken at several time points up to four hours.

ResultsPeak frequency and beat patterns are demonstrated for control and three reference compounds included in the EarlyTox Cardiotoxicity Kit as shown in Figures 2 through 5.

Figure 2. Untreated control iPSC cardiomyocytes

Figure 3. iPSC cardiomyocytes treated with propranolol exhibit a slower beat frequency

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Calcium signal change in untreated iPSC cardiomyocytes incubated for 2 hours with EarlyTox Cardiotoxicity Dye indicated by calcium peaks.

Propranolol is a non-selective blocker of 1- and 2-adrenergic receptors which inhibits the action of agonists of -adrenergic receptors. It slows peak frequency of calcium in iPSC cardiomyocytes.

0.82 M propranolol

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Figure 4. iPSC derived cardiomyocytes treated with isoproterenol show increased beat frequency

Isoproterenol is a non-selective -adrenergic agonist, which induces positive chronotropic and inotropic effects. It increases calcium peak frequency in iPSC cardiomyocytes.

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ency (BP

M)

Figure 5. iPSC cardiomyocytes treated with sotalol show decreased beat frequency and altered beat pattern

Sotalol is a non-selective competitive -adrenergic receptor blocker that also inhibits hERG. In addition to slowing peak frequency of calcium in iPSC cardiomyocytes, it alters the normal pattern.

1.23 M sotalol

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Log [sotalol] (M)

Beat frequ

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M)

Download Application Note:

Measuring Cardiac Activity: Intracellular Calcium Flux Detection on the FLIPR Tetra System

5moleculardevices.com/celltox

Drug-induced inhibition of the human ether--go-go-related gene (hERG) ion channel

has been related to the susceptibility of patients to a potentially fatal ventricular

tachyarrhythmia, torsade de pointes. In recent years, a number of FDA-approved

drugs were withdrawn from the market due to their off-target effect on hERG. As a

result, there has been an increasing need for identifying compounds that block the

hERG channel at earlier stages in the drug discovery process.

We studied the utility of our potassium assay kit on the FLIPR Tetra System to

investigate hERG compound activity. As presented in the application note below

(Characterization of hERG channel blockers using the FLIPR Potassium Assay Kit

on the FLIPR Tetra System) the assay exploits the permeability of thallium (Tl+) for

potassium (K+) channels, which is detected by a novel fluorescence indicator dye.

Seven reference hERG blockers are examined in a cell-based assay, and the results

are compared to values obtained using the IonWorks Barracuda Plus Automated

Patch Clamp System.

Resources

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Characterization of hERG channel blockers using the FLIPR Potassium Assay Kit on the FLIPR Tetra System

Download Application Note:

Characterization of hERG channel blockers using the FLIPR Potassium Assay Kit on the FlexStation 3 Multi-Mode Microplate Reader

Download Application Highlight:

Validation of the IonWorks Barracuda System for hERG Ion Channel Assay

Optimization of hERG channel stimulus buffer. Cells were incubated with dye and the buffers were added during detection on the FLIPR Tetra System. The concentration-dependent response of signal was characterized under different conditions. Optimal signal was obtained from the combination of 1mM Tl+ and 10 mM K+ (final concentration) stimulant buffer diluted in chloride-free buffer.

Concentration-dependent inhibition of hERG channel by reference compounds.

Benefits Functional measurement of K+

channel activity in a cell-based assay

Homogenous no-wash protocol reduces well-to-well variation and simplifies the workflow

Expanded signal window compared to non-homogenous assay

Cardiotoxicity

Characterization of hERG channel blockers

Figure 4. Electrophysiology assay on the IonWorks Barracuda System. Compounds were added at 3X final assay concentration in 1% DMSO and mixed with buffer in the well to achieve a 1X final compound concentration in 0.33% DMSO. Compounds were incubated for five minutes, after which a voltage protocol was applied with a one-second stimulation at +40 mV, followed by a one-second step to -50 mV for measurement of peak tail current.

Figure 3: Optimization of hERG channel stimulant. Cells were incubated with dye and the stimulant buffers were added during detection on the FLIPR Tetra System. The concentration-dependent response of signal was characterized under different conditions. Optimal signal was obtained from the combination of 1 mM Tl+ and 10 mM K+ (final concentration) stimulant buffer diluted in chloride-free buffer.

hE R G [ T l + ] = 0.5 mM

0 40 80 120 160

1.0

1.5

2.0

2.5Control10 mM K+

20 mM K+

Time

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asel

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hE R G [ T l + ] = 1 mM

0 40 80 120 160

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hE R G [ T l+ ] = 2 mM

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Experimental procedure Chinese hamster ovary (CHO) cells stably transfected with human Kv11.1 (hERG) ion channel were provided by ChanTest Corporation (Cleveland, OH). Cells are plated at 6,500/well in a 384-well, black-walled, clear bottom plate two days before the assay in growth medium including selection antibiotics and incubated at 37C and 5% CO2. Twenty four hours prior to the assay, the growth media is exchanged for induction media containing tetracycline. Four hours prior to the assay, the cells are switched from 37C to 28C for enhanced membrane expression of hERG channel. Plates are incubated with dye for one hour at room temperature in the dark.

For pharmacology analysis, hERG channel blocking compounds are added first and incubated for 30 minutes at room temperature. Previously optimized stimulus buffer is added to the well during detection on the FLIPR Tetra System. The 470-495 nm excitation LEDs and 515-575 nm emission filter are used). For comparison, parallel experiments were performed using the FluxOR Assay Kit (Life Technologies). The manufacturer assay protocol was followed when using this kit. Data is acquired at one second intervals for approximately 140 seconds. Data files are exported to GraphPad Prism for analysis.

Assay development is first carried out by determining optimal concentrations of thallium and potassium necessary for stimulation of the hERG channel. Plates are set up to test stimulating buffers containing various concentrations of K2SO4 and Tl2SO4.The Tl

+ and K+ buffers are diluted in 1X chloride-free buffer. Since thallium sulfate and potassium sulfate have two equivalents of cation per mole, they are considered as having 2X their respective cation concentrations. The stimulus buffers are added to cells during detection on the FLIPR Tetra System. Signal traces are compared among the concentration combinations to determine the optimal concentrations that provide the largest signal. Here, the optimal (final) Tl+ concentration that provided the largest signal for hERG is 1 mM and the optimal (final) K+ concentration is 10 mM (Figure 3).

Figure 2: FLIPR Potassium Assay Kit workflow on the FLIPR Tetra System.

Start with cells in 384-well plate

Add 20 L/well dye solution

If no media removal, incubate at RT/37C for

11.5 hrs

Add 10 L of 5X agonist

or antagonist

Incubate at RT or 37C for 30 min

Add 10 L of 6X potassium and thallium

stimulus buffer

Measure fluorescent

signals on FLIPR Tetra System

Figure 5. Concentration-dependent inhibition of hERG channel by reference compounds.

-10 -8 -6 -4 -20

1

2

3 CisaprideDofetilideTerfenadineHaloperidolPimozideFlunarizineAstemizole

L og [Inhibitor] M

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in)

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C24 Sweep 1C24 Sweep 2C24 Sweep 3C24 Sweep 4C24 Sweep 5C24 Sweep 1C24 Sweep 2C24 Sweep 3C24 Sweep 4C24 Sweep 5

C24

Voltage Protocol Negative Control Positive Control

Pre-additionPost-addition

Figure 4. Electrophysiology assay on the IonWorks Barracuda System. Compounds were added at 3X final assay concentration in 1% DMSO and mixed with buffer in the well to achieve a 1X final compound concentration in 0.33% DMSO. Compounds were incubated for five minutes, after which a voltage protocol was applied with a one-second stimulation at +40 mV, followed by a one-second step to -50 mV for measurement of peak tail current.

Figure 3: Optimization of hERG channel stimulant. Cells were incubated with dye and the stimulant buffers were added during detection on the FLIPR Tetra System. The concentration-dependent response of signal was characterized under different conditions. Optimal signal was obtained from the combination of 1 mM Tl+ and 10 mM K+ (final concentration) stimulant buffer diluted in chloride-free buffer.

hE R G [ T l+ ] = 0.5 mM

0 40 80 120 160

1.0

1.5

2.0

2.5Control10 mM K+

20 mM K+

Time

Res

pon

se: b

asel

ine

hE R G [ T l + ] = 1 mM

0 40 80 120 160

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1.5

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asel

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hE R G [ T l+ ] = 2 mM

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ine

Experimental procedure Chinese hamster ovary (CHO) cells stably transfected with human Kv11.1 (hERG) ion channel were provided by ChanTest Corporation (Cleveland, OH). Cells are plated at 6,500/well in a 384-well, black-walled, clear bottom plate two days before the assay in growth medium including selection antibiotics and incubated at 37C and 5% CO2. Twenty four hours prior to the assay, the growth media is exchanged for induction media containing tetracycline. Four hours prior to the assay, the cells are switched from 37C to 28C for enhanced membrane expression of hERG channel. Plates are incubated with dye for one hour at room temperature in the dark.

For pharmacology analysis, hERG channel blocking compounds are added first and incubated for 30 minutes at room temperature. Previously optimized stimulus buffer is added to the well during detection on the FLIPR Tetra System. The 470-495 nm excitation LEDs and 515-575 nm emission filter are used). For comparison, parallel experiments were performed using the FluxOR Assay Kit (Life Technologies). The manufacturer assay protocol was followed when using this kit. Data is acquired at one second intervals for approximately 140 seconds. Data files are exported to GraphPad Prism for analysis.

Assay development is first carried out by determining optimal concentrations of thallium and potassium necessary for stimulation of the hERG channel. Plates are set up to test stimulating buffers containing various concentrations of K2SO4 and Tl2SO4.The Tl

+ and K+ buffers are diluted in 1X chloride-free buffer. Since thallium sulfate and potassium sulfate have two equivalents of cation per mole, they are considered as having 2X their respective cation concentrations. The stimulus buffers are added to cells during detection on the FLIPR Tetra System. Signal traces are compared among the concentration combinations to determine the optimal concentrations that provide the largest signal. Here, the optimal (final) Tl+ concentration that provided the largest signal for hERG is 1 mM and the optimal (final) K+ concentration is 10 mM (Figure 3).

Figure 2: FLIPR Potassium Assay Kit workflow on the FLIPR Tetra System.

Start with cells in 384-well plate

Add 20 L/well dye solution

If no media removal, incubate at RT/37C for

11.5 hrs

Add 10 L of 5X agonist

or antagonist

Incubate at RT or 37C for 30 min

Add 10 L of 6X potassium and thallium

stimulus buffer

Measure fluorescent

signals on FLIPR Tetra System

Figure 5. Concentration-dependent inhibition of hERG channel by reference compounds.

-10 -8 -6 -4 -20

1

2

3 CisaprideDofetilideTerfenadineHaloperidolPimozideFlunarizineAstemizole

L og [Inhibitor] M

Res

pons

e: b

asel

ine

(max

- m

in)

0 1000 2000 3000

-50

0

50

Data 1

Time (ms)

Volta

ge (m

V)

0 1000 2000 3000-0.8

-0.4

0.0

0.4

0.8

1.2

Data2

Time (ms)

Cu

rren

t (n

A)

A02A02 Sweep 5A02 Sweep 4A02 Sweep 3A02 Sweep 2A02 Sweep 1A02 Sweep 5A02 Sweep 4A02 Sweep 3A02 Sweep 2A02 Sweep 10 1000 2000 3000

-0.8

-0.4

0.0

0.4

0.8

1.2

Data 1

Time (ms)

Cu

rren

t (n

A)

C24 Sweep 1C24 Sweep 2C24 Sweep 3C24 Sweep 4C24 Sweep 5C24 Sweep 1C24 Sweep 2C24 Sweep 3C24 Sweep 4C24 Sweep 5

C24

Voltage Protocol Negative Control Positive Control

Pre-additionPost-addition

Figure 4. Electrophysiology assay on the IonWorks Barracuda System. Compounds were added at 3X final assay concentration in 1% DMSO and mixed with buffer in the well to achieve a 1X final compound concentration in 0.33% DMSO. Compounds were incubated for five minutes, after which a voltage protocol was applied with a one-second stimulation at +40 mV, followed by a one-second step to -50 mV for measurement of peak tail current.

Figure 3: Optimization of hERG channel stimulant. Cells were incubated with dye and the stimulant buffers were added during detection on the FLIPR Tetra System. The concentration-dependent response of signal was characterized under different conditions. Optimal signal was obtained from the combination of 1 mM Tl+ and 10 mM K+ (final concentration) stimulant buffer diluted in chloride-free buffer.

hE R G [ T l+ ] = 0.5 mM

0 40 80 120 160

1.0

1.5

2.0

2.5Control10 mM K+

20 mM K+

Time

Res

pon

se: b

asel

ine

hE R G [ T l + ] = 1 mM

0 40 80 120 160

1.0

1.5

2.0

2.5

Time

Res

pon

se: b

asel

ine

hE R G [ T l+ ] = 2 mM

0 40 80 120 160

1.0

1.5

2.0

2.5

Time

Res

pon

se: b

asel

ine

Experimental procedure Chinese hamster ovary (CHO) cells stably transfected with human Kv11.1 (hERG) ion channel were provided by ChanTest Corporation (Cleveland, OH). Cells are plated at 6,500/well in a 384-well, black-walled, clear bottom plate two days before the assay in growth medium including selection antibiotics and incubated at 37C and 5% CO2. Twenty four hours prior to the assay, the growth media is exchanged for induction media containing tetracycline. Four hours prior to the assay, the cells are switched from 37C to 28C for enhanced membrane expression of hERG channel. Plates are incubated with dye for one hour at room temperature in the dark.

For pharmacology analysis, hERG channel blocking compounds are added first and incubated for 30 minutes at room temperature. Previously optimized stimulus buffer is added to the well during detection on the FLIPR Tetra System. The 470-495 nm excitation LEDs and 515-575 nm emission filter are used). For comparison, parallel experiments were performed using the FluxOR Assay Kit (Life Technologies). The manufacturer assay protocol was followed when using this kit. Data is acquired at one second intervals for approximately 140 seconds. Data files are exported to GraphPad Prism for analysis.

Assay development is first carried out by determining optimal concentrations of thallium and potassium necessary for stimulation of the hERG channel. Plates are set up to test stimulating buffers containing various concentrations of K2SO4 and Tl2SO4.The Tl

+ and K+ buffers are diluted in 1X chloride-free buffer. Since thallium sulfate and potassium sulfate have two equivalents of cation per mole, they are considered as having 2X their respective cation concentrations. The stimulus buffers are added to cells during detection on the FLIPR Tetra System. Signal traces are compared among the concentration combinations to determine the optimal concentrations that provide the largest signal. Here, the optimal (final) Tl+ concentration that provided the largest signal for hERG is 1 mM and the optimal (final) K+ concentration is 10 mM (Figure 3).

Figure 2: FLIPR Potassium Assay Kit workflow on the FLIPR Tetra System.

Start with cells in 384-well plate

Add 20 L/well dye solution

If no media removal, incubate at RT/37C for

11.5 hrs

Add 10 L of 5X agonist

or antagonist

Incubate at RT or 37C for 30 min

Add 10 L of 6X potassium and thallium

stimulus buffer

Measure fluorescent

signals on FLIPR Tetra System

Figure 5. Concentration-dependent inhibition of hERG channel by reference compounds.

-10 -8 -6 -4 -20

1

2

3 CisaprideDofetilideTerfenadineHaloperidolPimozideFlunarizineAstemizole

L og [Inhibitor] M

Res

pons

e: b

asel

ine

(max

- m

in)

0 1000 2000 3000

-50

0

50

Data 1

Time (ms)

Volta

ge (m

V)

0 1000 2000 3000-0.8

-0.4

0.0

0.4

0.8

1.2

Data2

Time (ms)

Cu

rren

t (n

A)

A02A02 Sweep 5A02 Sweep 4A02 Sweep 3A02 Sweep 2A02 Sweep 1A02 Sweep 5A02 Sweep 4A02 Sweep 3A02 Sweep 2A02 Sweep 10 1000 2000 3000

-0.8

-0.4

0.0

0.4

0.8

1.2

Data 1

Time (ms)

Cu

rren

t (n

A)

C24 Sweep 1C24 Sweep 2C24 Sweep 3C24 Sweep 4C24 Sweep 5C24 Sweep 1C24 Sweep 2C24 Sweep 3C24 Sweep 4C24 Sweep 5

C24

Voltage Protocol Negative Control Positive Control

Pre-additionPost-addition

Figure 4. Electrophysiology assay on the IonWorks Barracuda System. Compounds were added at 3X final assay concentration in 1% DMSO and mixed with buffer in the well to achieve a 1X final compound concentration in 0.33% DMSO. Compounds were incubated for five minutes, after which a voltage protocol was applied with a one-second stimulation at +40 mV, followed by a one-second step to -50 mV for measurement of peak tail current.

Figure 3: Optimization of hERG channel stimulant. Cells were incubated with dye and the stimulant buffers were added during detection on the FLIPR Tetra System. The concentration-dependent response of signal was characterized under different conditions. Optimal signal was obtained from the combination of 1 mM Tl+ and 10 mM K+ (final concentration) stimulant buffer diluted in chloride-free buffer.

hE R G [ T l+ ] = 0.5 mM

0 40 80 120 160

1.0

1.5

2.0

2.5Control10 mM K+

20 mM K+

Time

Res

pon

se: b

asel

ine

hE R G [ T l + ] = 1 mM

0 40 80 120 160

1.0

1.5

2.0

2.5

Time

Res

pon

se: b

asel

ine

hE R G [ T l+ ] = 2 mM

0 40 80 120 160

1.0

1.5

2.0

2.5

Time

Res

pon

se: b

asel

ine

Experimental procedure Chinese hamster ovary (CHO) cells stably transfected with human Kv11.1 (hERG) ion channel were provided by ChanTest Corporation (Cleveland, OH). Cells are plated at 6,500/well in a 384-well, black-walled, clear bottom plate two days before the assay in growth medium including selection antibiotics and incubated at 37C and 5% CO2. Twenty four hours prior to the assay, the growth media is exchanged for induction media containing tetracycline. Four hours prior to the assay, the cells are switched from 37C to 28C for enhanced membrane expression of hERG channel. Plates are incubated with dye for one hour at room temperature in the dark.

For pharmacology analysis, hERG channel blocking compounds are added first and incubated for 30 minutes at room temperature. Previously optimized stimulus buffer is added to the well during detection on the FLIPR Tetra System. The 470-495 nm excitation LEDs and 515-575 nm emission filter are used). For comparison, parallel experiments were performed using the FluxOR Assay Kit (Life Technologies). The manufacturer assay protocol was followed when using this kit. Data is acquired at one second intervals for approximately 140 seconds. Data files are exported to GraphPad Prism for analysis.

Assay development is first carried out by determining optimal concentrations of thallium and potassium necessary for stimulation of the hERG channel. Plates are set up to test stimulating buffers containing various concentrations of K2SO4 and Tl2SO4.The Tl

+ and K+ buffers are diluted in 1X chloride-free buffer. Since thallium sulfate and potassium sulfate have two equivalents of cation per mole, they are considered as having 2X their respective cation concentrations. The stimulus buffers are added to cells during detection on the FLIPR Tetra System. Signal traces are compared among the concentration combinations to determine the optimal concentrations that provide the largest signal. Here, the optimal (final) Tl+ concentration that provided the largest signal for hERG is 1 mM and the optimal (final) K+ concentration is 10 mM (Figure 3).

Figure 2: FLIPR Potassium Assay Kit workflow on the FLIPR Tetra System.

Start with cells in 384-well plate

Add 20 L/well dye solution

If no media removal, incubate at RT/37C for

11.5 hrs

Add 10 L of 5X agonist

or antagonist

Incubate at RT or 37C for 30 min

Add 10 L of 6X potassium and thallium

stimulus buffer

Measure fluorescent

signals on FLIPR Tetra System

Figure 5. Concentration-dependent inhibition of hERG channel by reference compounds.

-10 -8 -6 -4 -20

1

2

3 CisaprideDofetilideTerfenadineHaloperidolPimozideFlunarizineAstemizole

L og [Inhibitor] M

Res

pons

e: b

asel

ine

(max

- m

in)

0 1000 2000 3000

-50

0

50

Data 1

Time (ms)

Volta

ge (m

V)

0 1000 2000 3000-0.8

-0.4

0.0

0.4

0.8

1.2

Data2

Time (ms)

Cu

rren

t (n

A)

A02A02 Sweep 5A02 Sweep 4A02 Sweep 3A02 Sweep 2A02 Sweep 1A02 Sweep 5A02 Sweep 4A02 Sweep 3A02 Sweep 2A02 Sweep 10 1000 2000 3000

-0.8

-0.4

0.0

0.4

0.8

1.2

Data 1

Time (ms)

Cu

rren

t (n

A)

C24 Sweep 1C24 Sweep 2C24 Sweep 3C24 Sweep 4C24 Sweep 5C24 Sweep 1C24 Sweep 2C24 Sweep 3C24 Sweep 4C24 Sweep 5

C24

Voltage Protocol Negative Control Positive Control

Pre-additionPost-addition

Figure 4. Electrophysiology assay on the IonWorks Barracuda System. Compounds were added at 3X final assay concentration in 1% DMSO and mixed with buffer in the well to achieve a 1X final compound concentration in 0.33% DMSO. Compounds were incubated for five minutes, after which a voltage protocol was applied with a one-second stimulation at +40 mV, followed by a one-second step to -50 mV for measurement of peak tail current.

Figure 3: Optimization of hERG channel stimulant. Cells were incubated with dye and the stimulant buffers were added during detection on the FLIPR Tetra System. The concentration-dependent response of signal was characterized under different conditions. Optimal signal was obtained from the combination of 1 mM Tl+ and 10 mM K+ (final concentration) stimulant buffer diluted in chloride-free buffer.

hE R G [ T l+ ] = 0.5 mM

0 40 80 120 160

1.0

1.5

2.0

2.5Control10 mM K+

20 mM K+

Time

Res

pon

se: b

asel

ine

hE R G [ T l + ] = 1 mM

0 40 80 120 160

1.0

1.5

2.0

2.5

Time

Res

pon

se: b

asel

ine

hE R G [ T l+ ] = 2 mM

0 40 80 120 160

1.0

1.5

2.0

2.5

Time

Res

pon

se: b

asel

ine

Experimental procedure Chinese hamster ovary (CHO) cells stably transfected with human Kv11.1 (hERG) ion channel were provided by ChanTest Corporation (Cleveland, OH). Cells are plated at 6,500/well in a 384-well, black-walled, clear bottom plate two days before the assay in growth medium including selection antibiotics and incubated at 37C and 5% CO2. Twenty four hours prior to the assay, the growth media is exchanged for induction media containing tetracycline. Four hours prior to the assay, the cells are switched from 37C to 28C for enhanced membrane expression of hERG channel. Plates are incubated with dye for one hour at room temperature in the dark.

For pharmacology analysis, hERG channel blocking compounds are added first and incubated for 30 minutes at room temperature. Previously optimized stimulus buffer is added to the well during detection on the FLIPR Tetra System. The 470-495 nm excitation LEDs and 515-575 nm emission filter are used). For comparison, parallel experiments were performed using the FluxOR Assay Kit (Life Technologies). The manufacturer assay protocol was followed when using this kit. Data is acquired at one second intervals for approximately 140 seconds. Data files are exported to GraphPad Prism for analysis.

Assay development is first carried out by determining optimal concentrations of thallium and potassium necessary for stimulation of the hERG channel. Plates are set up to test stimulating buffers containing various concentrations of K2SO4 and Tl2SO4.The Tl

+ and K+ buffers are diluted in 1X chloride-free buffer. Since thallium sulfate and potassium sulfate have two equivalents of cation per mole, they are considered as having 2X their respective cation concentrations. The stimulus buffers are added to cells during detection on the FLIPR Tetra System. Signal traces are compared among the concentration combinations to determine the optimal concentrations that provide the largest signal. Here, the optimal (final) Tl+ concentration that provided the largest signal for hERG is 1 mM and the optimal (final) K+ concentration is 10 mM (Figure 3).

Figure 2: FLIPR Potassium Assay Kit workflow on the FLIPR Tetra System.

Start with cells in 384-well plate

Add 20 L/well dye solution

If no media removal, incubate at RT/37C for

11.5 hrs

Add 10 L of 5X agonist

or antagonist

Incubate at RT or 37C for 30 min

Add 10 L of 6X potassium and thallium

stimulus buffer

Measure fluorescent

signals on FLIPR Tetra System

Figure 5. Concentration-dependent inhibition of hERG channel by reference compounds.

-10 -8 -6 -4 -20

1

2

3 CisaprideDofetilideTerfenadineHaloperidolPimozideFlunarizineAstemizole

L og [Inhibitor] M

Res

pons

e: b

asel

ine

(max

- m

in)

0 1000 2000 3000

-50

0

50

Data 1

Time (ms)

Volta

ge (m

V)

0 1000 2000 3000-0.8

-0.4

0.0

0.4

0.8

1.2

Data2

Time (ms)

Cu

rren

t (n

A)

A02A02 Sweep 5A02 Sweep 4A02 Sweep 3A02 Sweep 2A02 Sweep 1A02 Sweep 5A02 Sweep 4A02 Sweep 3A02 Sweep 2A02 Sweep 10 1000 2000 3000

-0.8

-0.4

0.0

0.4

0.8

1.2

Data 1

Time (ms)

Cu

rren

t (n

A)

C24 Sweep 1C24 Sweep 2C24 Sweep 3C24 Sweep 4C24 Sweep 5C24 Sweep 1C24 Sweep 2C24 Sweep 3C24 Sweep 4C24 Sweep 5

C24

Voltage Protocol Negative Control Positive Control

Pre-additionPost-addition

6 moleculardevices.com/celltox

The EarlyTox Cell Integrity Kit is an optimized set of reagents that simplifies the

identification of live and dead cells. It can be used to measure the effects of different

treatments on cell viability and to evaluate toxic effects mediated through a variety

of mechanisms, including apoptosis and necrosis. This study shows how cytotoxicity

mediated through a variety of cellular mechanisms can easily be monitored and

quantified.

Used together with the SpectraMax MiniMax Imaging Cytometer and SoftMax Pro

Software, the EarlyTox Cell Integrity Kit offers a convenient way to determine live and

dead cell populations by fluorescence imaging.

Resources

Download Application Note:

Measuring cell viability and cytotoxicity with the EarlyTox Cell Integrity Kit on the SpectraMax MiniMax 300 Imaging Cytometer

Analysis of untreated and compound-treated HeLa cells. (A) Untreated cells (left panel) are mostly alive, with red fluorescent nuclei. At an intermediate concentration of compound (center panel), there is a mixture of live and dead cells. At high compound concentration (right panel), most cells are dead, with nuclei labeled both red and green. Images were acquired on the SpectraMax MiniMax 300 Imaging Cytometer. (B) Cells were identified as live (red masks) or dead (blue masks) using the Classification feature in SoftMax Pro Software.

HeLa cells were treated with anisomycin (red circles) or staurosporine (blue squares). Results were plotted using the 4-parameter curve fit in SoftMax Pro Software. IC50 for anisomycin was 3.3 M, and IC50 for staurosporine was 0.53 M; both values were consistent with published values.

Benefits Higher signal allows

shorter exposure times for fast results

Designed to work with many cell types

Streamlined image acquisition and analysis on a single system

Cellular Toxicity

Measuring cell viability and cytotoxicity

ResultsCells were imaged with the SpectraMax MiniMax 300 Imaging Cytometer, using green (541 nm emission) and red (713 nm emission) channels (Figure 2A). All cell nuclei in the images were automatically identified by setting size and threshold for object identification in the red channel. Cells were counted as live or dead using the Classification feature in the software, which can distinguish between nuclei that label red only vs. nuclei labeled red and green (Figure 2B). A detailed set of output parameters provided by the software includes percentage of live cells for each well. IC50 curves were plotted using the 4-parameter curve fit in SoftMax Pro Software (Figure 3).

ConclusionsUsed together with the SpectraMax MiniMax Imaging Cytometer, the EarlyTox Cell Integrity Kit offers a convenient way to determine live and dead cell populations by fluorescence imaging. Cytotoxicity mediated through a variety of cellular mechanisms can easily be monitored and quantified. SoftMax Pro Software distinguishes between live and dead cell staining patterns and plots the results automatically, saving time on analysis.

Figure 2. Analysis of untreated and compound-treated HeLa cells

A. Untreated cells (left panel) are mostly alive, with red fluorescent nuclei. At an intermediate concentration of compound (center panel), there is a mixture of live and dead cells. At high compound concentration (right panel), most cells are dead, with nuclei labeled both red and green. Images were acquired on the SpectraMax MiniMax 300 Imaging Cytometer.

B. Cells were identified as live (red masks) or dead (blue masks) using the Classification feature in SoftMax Pro Software.

Untreated 3.1 M Anisomycin 25 M Anisomycin

Figure 3. IC50 curves for cytotoxic compounds

HeLa cells were treated with anisomycin (red circles) or staurosporine (blue squares). Results were plotted using the 4-parameter curve fit in SoftMax Pro Software. IC50 for anisomycin was 3.3 M, and IC50 for staurosporine was 0.53 M; both values were consistent with published values.

Compound (M)

Perc

ent

Live

Cel

ls

A

B

ResultsCells were imaged with the SpectraMax MiniMax 300 Imaging Cytometer, using green (541 nm emission) and red (713 nm emission) channels (Figure 2A). All cell nuclei in the images were automatically identified by setting size and threshold for object identification in the red channel. Cells were counted as live or dead using the Classification feature in the software, which can distinguish between nuclei that label red only vs. nuclei labeled red and green (Figure 2B). A detailed set of output parameters provided by the software includes percentage of live cells for each well. IC50 curves were plotted using the 4-parameter curve fit in SoftMax Pro Software (Figure 3).

ConclusionsUsed together with the SpectraMax MiniMax Imaging Cytometer, the EarlyTox Cell Integrity Kit offers a convenient way to determine live and dead cell populations by fluorescence imaging. Cytotoxicity mediated through a variety of cellular mechanisms can easily be monitored and quantified. SoftMax Pro Software distinguishes between live and dead cell staining patterns and plots the results automatically, saving time on analysis.

Figure 2. Analysis of untreated and compound-treated HeLa cells

A. Untreated cells (left panel) are mostly alive, with red fluorescent nuclei. At an intermediate concentration of compound (center panel), there is a mixture of live and dead cells. At high compound concentration (right panel), most cells are dead, with nuclei labeled both red and green. Images were acquired on the SpectraMax MiniMax 300 Imaging Cytometer.

B. Cells were identified as live (red masks) or dead (blue masks) using the Classification feature in SoftMax Pro Software.

Untreated 3.1 M Anisomycin 25 M Anisomycin

Figure 3. IC50 curves for cytotoxic compounds

HeLa cells were treated with anisomycin (red circles) or staurosporine (blue squares). Results were plotted using the 4-parameter curve fit in SoftMax Pro Software. IC50 for anisomycin was 3.3 M, and IC50 for staurosporine was 0.53 M; both values were consistent with published values.

Compound (M)

Perc

ent

Live

Cel

ls

A

B

7moleculardevices.com/celltox

Drug-induced organ toxicity is an important cause of pharmaceutical candidates

failing to make it to market. Thus highly predictive assays for safety and efficacy

testing are crucial for improving drug development and reducing drug candidate

attrition. Human induced pluripotent stem cell (iPSC)-derived hepatocytes and

neurons, which exhibit typical characteristics and metabolism of mature cells, are

ideal for use in high-content screening in early drug development.

Although protocols for performing standard cytotoxicity assays using luminescence or

fluorescence readout on microplate readers are well known, even more information

can be gained by using an imaging cytometer to actually view the cells.

Viability dyes, such as Calcein AM, can be used to address gross compound toxicity

in live cells. Calcein AM only fluoresces green inside live cells that exhibit functional

esterase activity. iPSC-derived human hepatocytes were first treated with various

compounds for 24-hours then stained with Calcein AM. Images of live cells were

acquired using SpectraMax MiniMax 300 Imaging Cytometer, an upgradable option

for the SpectraMax i3 Multi-Mode Detection Platform. The green cytoplasmic area

of living cells was identified using the Cell Proliferation Protocol in SoftMax Pro

Software and IC50

values were determined using the softwares curve fit function.

Resources

Download Application Highlight:

Toxicity Assays Using Induced Pluripotent Stem Cell-Derived Cells

Download Poster:

Phenotypic in vitro Assessment of Compound Effects on Hepatotoxicity Using Novel Cell Models

Download Poster:

Phenotypic Assessment of Toxicity Using a Human Hepatocypte Co-Culture Model

Benefits Screen for toxicity in 96- or

384-well format easily and rapidly, using percentage of well area covered

Follow a familiar microplate reader workflow

Have confidence in the data output since the cells can be visualized

Toxicity assays using induced pluripotent stem cell-derived hepatocytes

A viability dye allows live cell measurement. Live iPSC-derived hepatocytes stained with Calcein AM are identified with a SoftMax Pro Software Cell Proliferation Protocol. This assay is used to calculate the percentage of wells covered by live cells. The purple masks on the right show segmentation of live cells which are visible in the images on the left.

BenefitsDrug-induced organ toxicity is an important cause of pharmaceutical candidates failing to make it to market. Thus highly predictive assays for safety and efficacy testing are crucial for improving drug development and reducing drug candidate attrition. Human induced pluripotent stem cell (iPSC)-derived hepatocytes and neurons, which exhibit typical characteristics and metabolism of mature cells, are ideal for use in high content screening in early drug development.

Although protocols for performing standard cytotoxicity assays using luminescence or fluorescence readout on microplate readers are well-known, even more information can be gained by using an imaging cytometer to actually view the cells.

Getting more information from a standard viability assayViability dyes, such as Calcein AM, can be used to address gross compound toxicity in live cells. Calcein AM only fluoresces green inside live cells that exhibit functional esterase activity. iPSC-derived human hepatocytes were first treated with various compounds for 24-hours then stained with Calcein AM. Images of live cells were acquired using SpectraMax MiniMax Imaging Cytometer, an upgradable option for the SpectraMaxi3 Multi-Mode Detection Platform. The green cytoplasmic area of living cells was identified using the Cell Proliferation Protocol in SoftMax Pro Software (Figures 1 and 2) and IC50 values were determined using the softwares curve fit function (Figure 3).

Screen for toxicity in 96- or384-well format easily andrapidly, using percentage ofwell area covered

Follow a familiar microplatereader workflow

Have confidence in thedata output since the cellscan be visualized

Figure 1. A viability dye allows live cell measurement Figure 2. Determine live cell confluency at a glance

Live cells stained with Calcein AM are identified with a SoftMax Pro Software Cell Proliferation Protocol. This assay is used to calculate the percentage of wells covered by live cells. The purple masks on the right show segmentation of live cells which are visible in the images on the left.

In a hepatotoxicity experiment, a heat map of the results allows you to quickly determine which compounds are toxic. Red wells are the most confluent and blue wells are the least, showing highest toxicity in rows E-H and empty wells in rows A, MP.

Toxicity assays using induced pluripotent stem cell-derived cells

Figure 3. Compound IC50 values determined from curve-fit

IC50 values for four different compounds were determined using the curve fit function of SoftMax Pro Software.

Hepatotoxicity

8 moleculardevices.com/celltox

Development of quantitative high-throughput in vitro assays that enable assessment

of viability and morphological changes in neuronal cells is an active area of

investigation in drug discovery and environmental chemical safety assessment. High-

content imaging is an emerging and efficient tool for generating multidimensional

quantitative cellular readouts; in addition, human iPSC-derived neurons are a

promising in vitro model system that emulates both the functionality and behavior

of mature neurons, and they are available in quantities sufficient for screening

workflows.

The goal of this study was to develop high-content imaging and analysis methods to

assess multiple phenotypes in human iPSC-derived neuronal cells. Specifically, cell

culture, staining, and imaging protocols were optimized in a 384-well assay format

and laboratory workflow was improved by designing a one-step procedure to reduce

assay time and minimize cell disturbance. Phenotypic readouts include quantitative

characterization of neurite outgrowth and branching, cell number and viability, as well

as measures of adverse effects on mitochondrial integrity and membrane potential.

To verify the robustness of the workflow, a series of established toxicant compounds

were tested. Concentration response effects of selected test compounds on human

iPSC-derived neuronal cells were measured and illustrate how the proposed methods

may be used for high-content high-throughput compound toxicity screening and

safety evaluation of drugs and environmental chemicals.

Resources

Download Article Reprint:

High-Content High-Throughput Assays for Characterizing the Viability and Morphology of Human iPSC-Derived Neuronal Cultures

Download Poster:

In vitro of Neurotoxicity Hazards of Environmental Compounds Using Human iPSC-Derived Neurons

Benefits Neuronal bodies and

processes can be visualized simultaneously in live cells

Assess both the morphological features of cultured neurons and the extent and degree of complexity of neuronal networks

Neurotoxicity

Characterizing the viability and morphology of human iPSC-derived neuronal cultures

iCell Neurons were cultured in 384-well plates for 5 days at a density of 7,000 cells/ well and visualized live with Calcein AM. Left panel shows representative fluorescence microscopy images us-ing either 10 (top row), or 20 (bottom row) objectives. Right panel shows the outcome of the automated identification of the neuronal bodies (red dots) and outgrowth (thin red lines) using the Neurite Out-growth module in the MetaXpress 5 Software.

objective. The light source was a solid state white light engine with

emission from 380nm to 680nm. A FITC filter cube [Ex 482/35, Em

536/40; center wavelength (nm)/bandpass width (nm)] was used for

Calcein AM. A DAPI filter cube (Ex 377/50, Em 447/60) was used

for Hoechst 33342.

Images for the mitochondrial permeability assay were acquired

using the TRITC and FITC channels for the JC-10 staining along with

a DAPI channel if the Hoechst 33342 staining was included. For

higher throughput with the multiparametric toxicity assays, one

image per well was taken for 384-well plates using a 10 objective.Images acquired with a 20 objective allowed higher resolution,better visualization, and improved analysis of the mitochondria.

Images were analyzed using theMetaXpress 5 software (Molecular

Devices) Granularity and Neurite Outgrowth modules with or without

single cell segmentation based on the optional nuclear staining. Sta-

tistical analysis of the data included calculation of a Z0-factor coeffi-cient. As used in this study, it represents the

separation of positive or high concentration

and negative control wells on a given plate.

Typically, eight control wells each for positive

and negative controls were included on each

plate and Z0-factor was calculated using thefollowing formula: Z0 =1[(3*spos+3*sneg)/j(MposMneg)j], where s= standard deviationand M = average value. EC50 values weredetermined using the 4-parameter curve fit

from the SoftMax Pro software (Molecular

Devices).

RESULTSDevelopment and Optimizationof the Live-Cell High-ContentAssay with iPSC-Derived Neurons

The primary goal of this study was to

develop and evaluate fast, accurate, and re-

producible high-content and high-throughput

methods to investigate effects of test com-

pounds on the morphology and viability of

iPSC-derived neurons using live and fixed

cells. During development, neurons become

assembled into functional networks by ex-

tending the outward growth of axons and

dendrites (collectively called neurites) that

connect synaptically to other neurons.

Traditional markers that are widely used in

in vitro studies of neuronal cell viability and

neurite outgrowth are b-III tubulin andmicrotubule-associated protein 2 (MAP2).

Class III b-tubulin, or b-III tubulin, is amicrotubule element of the tubulin family

found almost exclusively in neurons.28,29

Monoclonal antibodies against b-III tubulinare traditionally used to identify neurons

in samples of brain tissue or primary preps of neurons, separat-

ing neurons from glial cells. MAP2 belongs to the microtubule-

associated protein family and is thought to be involved in microtubule

assembly, which is an essential step in neurogenesis. MAP2 serves

to stabilize microtubule growth by crosslinking of microtu-

bules with each other and other intermediate filaments.28,29 While

b-III tubulin and MAP2 provide neuronal cell specificity to thein vitro assay readouts, these assays are time consuming, lower-

throughput, and involve multiple steps, including cell fixation,

permeabilization, blocking, antibody incubations, and washing.

Each of these steps has the potential to disrupt the neuronal cells

and introduce variability. Therefore, we tested whether Calcein AM,

a homogeneous cell-permeant dye that can be used to determine

cell viability in most eukaryotic cells, alone, or in combination

with a nuclear stain (e.g., Hoechst 33342), would streamline data

acquisition and analysis (Fig. 1).

Fig. 1. iCell Neurons were cultured in 384-well plates for 5 days at a density of 7,000 cells/well and visualized live with Calcein AM. Left panel shows representative fluorescence mi-croscopy images using either 10 (top row), or 20 (bottom row) objectives. Right panelshows the outcome of the automated identification of the neuronal bodies (red dots) andoutgrowth (thin red lines) using the Neurite Outgrowth module in the MetaXpress 5 software.

SIRENKO ET AL.

538 ASSAY and Drug Development Technologies NOVEMBER/DECEMBER 2014

9moleculardevices.com/celltox

Autophagy is an intracellular catabolic process that sequesters and degrades

proteins and organelles that have either been recognized as faulty or are simply

no longer needed by the cell. Autophagy supports normal cellular processes by

carrying out the housekeeping function of removing invasive microorganisms,

misfolded proteins, and degraded organelles. Autophagy is essential to the

maintenance of cell homeostasis and, in addition to its role as a mechanism of cell

material turnover, autophagy supports the response of organisms to challenges such

as starvation by adjusting rates of energy consumption and material re-utilization.

Both beneficial and harmful roles for autophagy have been discovered in cancer,

infectious disease, diabetes, neurodegenerative disease, and other conditions. The

role of autophagy is of particular interest in the study of neuronal cells, where it helps

to maintain protein structure and function over long axonal distances. Unlike the cells

of other tissues, the cytosolic contents of neurons are not routinely diluted by mitosis.

Altered autophagy and accumulation of cellular toxins are seen in Alzheimers

disease, Parkinsons disease, and other neurological disorders. Quantitation of

the effects of experimental variables on this process is an essential element of

research that may lead to the development of safe and effective treatments targeting

autophagy.

Resources

Download Application Note:

Visualizing Subcellular Vessicles to Quantitate Autophagy in Neuronal Cells

Download Poster:

Enhanced Imaging for Accurate Quantification of Autophagy in Neuronal Cells

Benefits Accurate segmentation of

unlabeled neurites

Deeper insights into mechanisms of neurodegenerative disease

Precise discrimination of similar subcellular structures

Rich data generated by measuring multiple biologies in one well

Autophagy

Visualizing subcellular vesicles to quantitate autophagy in neuronal cells

Measuring autophagosome response to various compounds: Representative 60X magnification images of rat PC-12 cells following exposure to three different compounds at three different concentrations. Autophagosomes (green) can be clearly visualized and measured.

Measuring autophagosome response to various compounds: Dose-response effect of chloroquine, rotenone, and verapamil on ag-gregate autophagosome area in rat PC-12 cells. Autophagosomes may be inhibited or stimulated by compound exposure.

2

Figure 2: The straightforward and automated workflow is amenable to high-throughput screening with minimal hands-on time required even for an experiment that may take days from plating to end results.

Figure 3: (Left) Representative 60X magnification images of human neurons following exposure to three different compounds at three different concentrations. Autophagosomes (green) respond to the cell treatment in a dose-dependent manner. (Right) Dose-response effect of chloroquine, rotenone, and verapamil on aggregate autophagosome area in human neurons. Autophagosomes may be inhibited or stimulated by compound exposure.

Quantitationmethods The level of Cyto-ID Green staining of autophagosomes and LysoTracker Deep Red staining of lysosomes in live cells provides information about the mechanism of a compounds stimulation or inhibition of the autophagy pathway. This can be accomplished using the ImageXpress Micro XLS System, a high-content screening system for widefield fluorescence or brightfield imaging of fixed or live cells, tissues, and small organisms.

MetaXpress software analyzes images using either pre-configured applications or customized modules to characterize phenotypic changes and produce specific outputs. A Custom Module enables the simultaneous detection, measurement and area summation of multiple stained bodies, such as nuclei, autophagosomes, and lysosomes. Measured values can be plotted over time or against experimental conditions such as a range of drug compound concentrations.

Increasedquantityofautophagosomesindicatescelldistress In the examples that follow, iCell (Cellular Dynamics Intl.) human-induced pluripotent stem cell (iPSC) derived neurons and rat PC-12 cells were used to demonstrate the utility of testing for multiple cellular responses in each well of a 384-well microplate.

HumanneuronsNeurons were plated at 5000 cells/well and grown as following the manufacturers recommendations. After 5 days, cells were treated with a dilution series of compounds prepared in maintenance media. After 24 hours of compound incubation, staining for lysosomes, nuclei and autophagosomes was performed and live cells were imaged using ImageXpress Micro XLS Widefield Automated System with the chamber heated to 37C. Images were acquired from 2-4 sites per well using either a 60X Plan Fluor or 40X Plan Apo objective. Image analysis was accomplished using MetaXpress software to quantitate the effects of exposure to experimental compounds over a range of concentrations. While example images show cells at 60X magnification for illustrative purposes, the data in the graphs in Figures 3-6 are from the

Compound(M)

Autop

hagos

ometotalarea/ce

ll

Compound(M)

Autop

hago

sometotalarea/ce

ll

Figure 4: (Left) Representative 60X magnification images of rat PC-12 cells following exposure to three different compounds at three different concentrations. Autophagosomes (green) can be clearly visualized and measured. (Right) Dose-response effect of chloroquine, rotenone, and verapamil on aggregate autophagosome area in rat PC-12 cells. Autophagosomes may be inhibited or stimulated by compound exposure.

Day 0Thaw&

platecells

Day 5Add

compounds

Day 6Stain,acquire

images,analyze

Chloroquine Verapamil Rotenone

60M 60M 3.33M

7.50M 7.50M 0.001M

0.47M 0.47M 0.002M

Chloroquine Verapamil Rotenone

30M 30M 0.37M

0.37M 0.37M 0.04M

0.04M 0.01M 0.01M

2

Figure 2: The straightforward and automated workflow is amenable to high-throughput screening with minimal hands-on time required even for an experiment that may take days from plating to end results.

Figure 3: (Left) Representative 60X magnification images of human neurons following exposure to three different compounds at three different concentrations. Autophagosomes (green) respond to the cell treatment in a dose-dependent manner. (Right) Dose-response effect of chloroquine, rotenone, and verapamil on aggregate autophagosome area in human neurons. Autophagosomes may be inhibited or stimulated by compound exposure.

Quantitationmethods The level of Cyto-ID Green staining of autophagosomes and LysoTracker Deep Red staining of lysosomes in live cells provides information about the mechanism of a compounds stimulation or inhibition of the autophagy pathway. This can be accomplished using the ImageXpress Micro XLS System, a high-content screening system for widefield fluorescence or brightfield imaging of fixed or live cells, tissues, and small organisms.

MetaXpress software analyzes images using either pre-configured applications or customized modules to characterize phenotypic changes and produce specific outputs. A Custom Module enables the simultaneous detection, measurement and area summation of multiple stained bodies, such as nuclei, autophagosomes, and lysosomes. Measured values can be plotted over time or against experimental conditions such as a range of drug compound concentrations.

Increasedquantityofautophagosomesindicatescelldistress In the examples that follow, iCell (Cellular Dynamics Intl.) human-induced pluripotent stem cell (iPSC) derived neurons and rat PC-12 cells were used to demonstrate the utility of testing for multiple cellular responses in each well of a 384-well microplate.

HumanneuronsNeurons were plated at 5000 cells/well and grown as following the manufacturers recommendations. After 5 days, cells were treated with a dilution series of compounds prepared in maintenance media. After 24 hours of compound incubation, staining for lysosomes, nuclei and autophagosomes was performed and live cells were imaged using ImageXpress Micro XLS Widefield Automated System with the chamber heated to 37C. Images were acquired from 2-4 sites per well using either a 60X Plan Fluor or 40X Plan Apo objective. Image analysis was accomplished using MetaXpress software to quantitate the effects of exposure to experimental compounds over a range of concentrations. While example images show cells at 60X magnification for illustrative purposes, the data in the graphs in Figures 3-6 are from the

Compound(M)

Autop

hago

sometotalarea/ce

ll

Compound(M)

Autop

hago

sometotalarea/ce

ll

Figure 4: (Left) Representative 60X magnification images of rat PC-12 cells following exposure to three different compounds at three different concentrations. Autophagosomes (green) can be clearly visualized and measured. (Right) Dose-response effect of chloroquine, rotenone, and verapamil on aggregate autophagosome area in rat PC-12 cells. Autophagosomes may be inhibited or stimulated by compound exposure.

Day 0Thaw&

platecells

Day 5Add

compounds

Day 6Stain,acquire

images,analyze

Chloroquine Verapamil Rotenone

60M 60M 3.33M

7.50M 7.50M 0.001M

0.47M 0.47M 0.002M

Chloroquine Verapamil Rotenone

30M 30M 0.37M

0.37M 0.37M 0.04M

0.04M 0.01M 0.01M

10 moleculardevices.com/celltox

A large number of environmental agents, with potential for human exposure,

remain inadequately tested for toxicological effects. Hence, there is a pressing

need to develop a high-throughput in vitro battery of assays to rapidly screen

these compounds to facilitate prioritization for more comprehensive testing in

vivo. Amongst drugs, cardiotoxicity is a leading cause of compound attrition during

preclinical drug development. Cardiotoxicity arises from interference with normal

cellular electrophysiology and contractility as well as biochemical pathways. New

technologies have emerged to screen for adverse events that fall within the scope

of cardiotoxicity. The extent to which environmental compounds contribute to

cardiotoxicity remains largely unknown.

As part of an effort to develop and characterize in vitro model systems for

toxicological screening, we used fast kinetic fluorescence imaging that monitored

changes in intracellular Ca2+ using a calcium-sensitive dye to screen a library of

environmental chemicals and drugs for their ability to alter cellular viability, beating

rates, and beating patterns in human iPSC-derived cardiomyocytes. The assay was

optimized for high-throughput screening and characterizes beating profiles by using

multi-parameter analysis outputs such as beating rate, peak frequency and width,

amplitude, or waveform irregularities.

Resources

Download Poster:

In vitro Assessment of Cardiotoxicity Hazard of Environmental Compounds Using Fast Fluorescence Imaging of Beating iPSC-Derived Cardiomyocytes

Summary An assay model is presented

for the in vitro assessment of cardiotoxicity hazards that is well-suited for automated screening of environmental chemicals.

The assay system is amenable to concentration-response modeling and can be used to prioritize suspected cardiotoxicants

Flame retardants and pesticides appear to be more toxic at 30 min compared to PAHs and drugs.

Case Study

In vitro assessment of cardiotoxicity hazard of environmental compoundsDivision of the National Toxicology Program, National Institute of Environmental Health Services

2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

In vitro Assessment of Cardiotoxicity Hazard of Environmental Compounds Using Fast Fluorescence

Imaging of Beating iPSC-Derived Cardiomyocytes

Sirenko O1, Ryan KR

2, Behl M

2,3, Parham F

2, Anson B

4, Cromwell EF

1, Tice RR

2

1. Molecular Devices, LLC, Sunnyvale, CA 2. Division of the National Toxicology Program, NIEHS/NIH, RTP, NC 3. Kelly Government Solutions, RTP, NC 4. Cellular Dynamics International, Madison, WI

Introduction

A large number of environmental agents, with potential for human exposure, remain inadequately tested for

toxicological effects. Hence, there is a pressing need to develop a high-throughput in vitro battery of assays to

rapidly screen these compounds to facilitate prioritization for more comprehensive testing in vivo. Amongst

drugs, cardiotoxicity is a leading cause of compound attrition during preclinical drug development.

Cardiotoxicity arises from interference with normal cellular electrophysiology and contractility as well as

biochemical pathways. New technologies have emerged to screen for adverse events that fall within the scope

of cardiotoxicity. The extent to which environmental compounds contribute to cardiotoxicity remains largely

unknown. As part of an effort to develop and characterize in vitro model systems for toxicological screening,

we used fast kinetic fluorescence imaging that monitored changes in intracellular Ca2+using a calcium sensitive

dye to screen a library of environmental chemicals and drugs for their ability to alter cellular viability, beating

rates, and beating patterns in human iPSC-derived cardiomyocytes. The assay was optimized for high

throughput screening and characterizes beating profiles by using multi-parameter analysis outputs such as

beating rate, peak frequency and width, amplitude, or waveform irregularities.

Figure 1. Cartoon of Ca2+

flux in cardiomyocyte

contractions. 1. Membrane depolarization occurs &

Ca2+enters cytosol. 2. Calcium Induce Calcium Release

(CIRC) intracellular Ca2+

is released from

sarcoplasmic reticulum. 3. Cytoplasmic Ca2+

binds to

troponin, activates sarcomere. 4. Cardiomyocyte

contraction occurs. 5. Removal of Ca2+

by active

transport into the sarcoplasmic reticulum and Ca2+

exchange with extracellular fluid. 6. Cycle repeats.

iPSC derived iCellCardiomyocytes

Cells were received frozen from Cellular Dynamics International (CDI). Cells were thawed

and plated according to CDI protocol and incubated for 14 days. The presence of strong

synchronous contractions of cells in the wells under the light microscope was confirmed

prior to running experiments.

FLIPR Tetra System Ca2+Flux Assay

Experimental plates were loaded and a pre-drug read was acquired. Acquisition was set

up for EarlyTox Cardiotoxicity dye (Ex 485 nm, Em 530 nm). Data was acquired at ~8 fps.

Data was analyzed using FLIPR Screenworks Peak Pro software to automatically detect

and characterize individual contraction events. GraphPad Prism was used to plot the

curves.

Figure 2. Top: Images of cardiomyocytes

going through one contraction cycle.

Cells were loaded with EarlyTox

Cardiotoxicity dye and imaged on

ImageXpress Micro XL system with a

20x objective. Intensity is proportional

to local Ca2+

concentration. Left:

Graphical representation of parameters

automatically measured by FLIPR

Screenworks Peak Pro Software.

In vitro Cardiotoxicity Assay

EarlyTox Cardiotoxicity Kit for Ca2+Flux Assay

The EarlyTox Cardiotoxicity Kit is designed to work with stem cell derived cardiomyocytes

or primary cardiomyocytes.

Bench Mark Concentration Analysis

Dose-response information is critical for ranking compounds for their toxicity or safety. However, IC50/EC

50analysis is limited

by the need for high/low asymptotes. In toxicity assessment, high concentration response plateaus are often not present,

leading to a need for an improved analysis method. To overcome this limitation, we applied the Benchmark Concentration

(BMC) analysis method developed by EPA for analysis of toxicity testing2,3

to the multi-parametric beating cardiomyocyte

assay. A number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width,

raise, decay) and regularity were collected. Viability was examined at 60 min and 24 hrs using the number of Calcein AM

positive cells. The cutoff for viability was set at 3 standard deviations below the DMSO mean. Data points with viability

below the cutoff were omitted from BMC analysis.

Am

plit

ude

Spacing or BPM

Peak Width

(10% Amp)

Rise &

Decay

times

Toxicity Library Screening

A diverse set of 80 environmental chemicals (e.g., flame retardants, PAHs, pesticides, mitochondrial toxicants) and drugs

were screened for cardiotoxicity acro