Figure 1: HeLa cells expressing NucLight Red. (Left) Untreated HeLa NucLight

Red cells. (Right) HeLa NucLight Red cells treated with 300 nM staurosporine.

Blended phase-contrast and red/green images taken at 20x illustrate homogenous

express of red nuclear signal and activation of caspase-3/7 (green). HD phase

images provide a qualitative assessment of morphological correlates to apoptosis

(left).

Introduction

Apoptosis, the biological process by which cells undergo

programmed cell death, is required for normal tissue

maintenance and development. However, aberrations in

apoptotic signaling networks are implicated in numerous

human diseases including neurodegeneration and cancer[1].

Apoptotic pathways are initiated by extrinsic factors that result

in activation of pro-apoptotic receptors on the cell surface, or

intrinsically by many different stimuli such as DNA damage,

hypoxia, the absence of growth factors, defective cell cycle

control, or other types of cellular stress that result in release of

cytochrome C from mitochondria.

Stimulation of either the extrinsic or intrinsic apoptotic

pathways triggers a signaling cascade that results in the

activation of a family of proteins that play a major role in

carrying out the apoptotic process called caspases[2].

Caspases (cysteinyl aspartate proteinases) cleave substrates

following an Asp (D) amino acid residue. Effector targets of

caspases include caspase family members themselves, proteins

involved in fragmentation of cellular DNA (Caspase Activated

DNAses), nuclear lamins, as well as proteins that make up the

cell cytoskeleton. Caspase proteins are traditionally separated

into two groups, initiator caspases (caspase 2, 8, 9 and 10),

and executioner or effector caspases (caspase 3, 6, and 7). As

a primary executioner caspase in most systems, the activation

of caspase-3 often results in the irreversible commitment of a

cell to apoptosis. Therefore, the activation of caspase-3 is

considered a reliable marker for cells undergoing apoptosis.

Numerous in vitro assays have been designed to measure the

activation of caspase-3. The majority of these assays utilize

reagent substrates that incorporate the DEVD (Asp-Glu-Val-

Asp) motif which is recognized by both activated caspases 3

and 7[3]. This motif has been incorporated into luciferase,

colorimetric, and fluorometric substrates that can be used in a

variety of assay types, all of which result in only a single,

user-defined time point measurement of caspase-3/7 activity.

In addition, these techniques require multiple wash steps or

cell lifting prior to data collection; potentially resulting in the

loss of cells or critical data in experiments where cells

undergo apoptosis at different rates according to treatment

conditions.

Following the work of Daya et.al.[4], we introduce an

optimized assay system incorporating the Essen CellPlayer™

Kinetic Caspase-3/7 Apoptosis reagent (kinetic apoptosis

reagent) for use on the IncuCyte FLR™ or ZOOM™ imaging

systems. When added to the tissue culture growth medium,

this inert, non-fluorescent substrate freely crosses the cell

membrane where it is cleaved by activated caspase-3/7

resulting in the release of the DNA dye and green fluorescent

labeling of DNA[5]. In addition, Essen’s lentivirus based

CellPlayer NucLight Red reagent, used to reliably label cell

nuclei in a nonperturbing way, provides a means to kinetically

quantify cell proliferation over time (Figure 1). In

combination, the caspase-3/7 reagent and NucLight label

provide a multiplexed way to differentiate inhibition of cell

growth and induction of cell death. High definition phase

contrast images provide an additional qualitative validation of

cell death based on morphological characteristics. Finally,

using any number of strategies (e.g. area under the curve, max

counts, single time points), the kinetic data generated using

this assay strategy can be used to derive informed

pharmacology measurements.

Approach and Methods

Cell culture and assay procedure

Prior to beginning the assay, cells were grown to confluence in

25 cm2 tissue culture-treated flasks. MDA-MB-231, HeLa,

A549, and MCF-7 cells were cultured in F12-K (Gibco)

CellPlayer™ 96-Well Kinetic Caspase-3/7 Apoptosis Assay Katherine Artymovich, Thom Nelson, Tim Dale, Susana Alcantara, Eric Endsley, Daniel M. Appledorn,

Del Trezise, Vince Groppi Essen BioScience – Ann Arbor, Michigan – Welwyn Garden City, UK

CellPlayer™ 96-Well Kinetic Caspase-3/7 Apoptosis Assay

2

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Figure 2: Staurosporine (SSP) induced caspase-3/7 activity in human breast adenocarcinoma cells (MDA-MB-231). (A) Representative phase contrast and fluorescent images reveal classical apoptotic cell morphologies and indicate activation of caspase-3/7, respectively. The customizable object counting algorithm identifies fluorescent objects as indicated with red Xs (IncuCyte FLR). (B) Kinetic measures of the number of caspase-3/7 positive cells is recorded over time and plotted as fluorescent objects, n=3 wells per data point shown (C) At the 48 hour end point, the apoptotic index was calculated by dividing the number of Caspase-3/7 fluorescent objects by the total number of DNA containing objects following staining with Vybrant DyeCycle Green.

supplemented with Pen-Strep, 10% FBS, and 2 mM

GlutaMAX (Gibco). HUVECs were cultured in complete

Lonza EGM-2 BulletKit and were grown no further than

passage 6. HeLa cells were infected with Essen’s CellPlayer

NucLight Red (Lenti, EF1a, puromycin) reagent (MOI of 3

TU/cell). Positively expressing cells were selected for in

complete media containing 1µg/ml puromycin for 24 hours

and then maintained in 0.5µg/ml puromycin. The day before

starting the assay, cells were plated at 2500 cells/well

(HUVEC) or 5000 cells/well (MDA-MB-231, HeLa NucLight

Red, A549, and MCF-7) in a 96-well plate. Cells were

allowed to adhere and grow overnight so that they were at

~25-35% confluence at the start of the assay. Prior to addition

of the kinetic apoptosis reagent and/or treatment conditions,

HUVECs were starved for two hours in 0.2% serum with no

additional growth factors. Staurosporine (SSP) or Taxol were

serially diluted with F12-K growth medium (100µl per well)

containing the kinetic apoptosis reagent at a final

concentration of 5µM. Transcription necrosis factor alpha

(TNF-α) was serially diluted in F12-K growth medium (100µl

per well) containing 5µg/ml cycloheximide and 5µM kinetic

apoptosis reagent. DMSO did not exceed 0.7% and did not

affect proliferation or cell morphology relative to complete

medium (data not shown). Cells were placed in an IncuCyte

FLR or ZOOM with a 10X objective in a standard cell culture

incubator at 37°C and 5% CO2. Two images per well were

collected every 2-3 hours in both phase-contrast and

fluorescence. The assay was considered complete when a

maximal response was achieved as determined by image

analysis

Data quantification and analysis

Throughout the assay, both phase and fluorescent images were

collected, detecting both morphological hallmarks of apoptosis

and caspase-3/7 activity, respectively. The integrated object

counting algorithm was used to isolate the fluorescent nuclear

signal from background, segment the signal into individual

objects, and count objects on a per area basis for each time

point. Because this reagent labels DNA and it is known that

nuclear fragmentation is a hallmark of apoptosis, in many

cases there is not a linear relationship of cell nuclei to counted

objects. As a result, we have also successfully used the object

confluence metric (the percentage of the image occupied by

fluorescent objects) to kinetically quantify caspase-3/7 activity

in this assay (see additional technical note). Both object count

and object confluence metrics result in similar kinetic curves.

Therefore, object count is presented throughout this

application note. As an additional marker of proliferation, and

to correct for differential proliferation of cells in one color

(green) experiments, the total number of DNA containing

objects was counted at the final time point using Vybrant

Green. This number was used to calculate the “apoptotic

index”, defined as the number of caspase-3/7 positive objects

divided by the total number of DNA containing objects.

Results and Discussion

The kinetic activation of Caspase-3/7 can be quantitatively

measured using the Essen CellPlayer Kinetic Caspase-3/7

Apoptosis reagent in the IncuCyte FLR and ZOOM

The first experiment using the kinetic apoptosis reagent was

designed to illustrate our ability to detect cells with activated

caspase-3/7 using a well-known inducer of apoptosis, the

CellPlayer™ 96-Well Kinetic Caspase-3/7 Apoptosis Assay

3

Figure 3: Pharmacological analysis of caspase-3/7 activation and nuclear counts

in HeLa NucLight Red cells treated with SSP. (A) Blended phase-contrast and

red/green images taken at 20x show red nuclear signal and activation of caspase-3/7

as well as morphological differences in untreated cells (left) vs. cells treated with

300nM SSP (right). (B) Caspase-3/7 positive objects and (C) nuclear counts were

measured over time in response to increasing concentrations of SSP. (D) Area under

the curve (AUC) of nuclear counts/mm2 and caspase object counts/mm2 over time

were measured and used to calculate IC50 and EC50 values, respectively.

general protein kinase inhibitor staurosporine (SSP). To

accomplish this, we treated MDA-MB-231 cells, a human

breast adenocarcinoma derived cell line with SSP serially

diluted in growth media containing 5 µM kinetic apoptosis

reagent in a 96-well plate. Once treated, the cells were

immediately placed inside the IncuCyte FLR imaging platform

with a 10X objective in a standard cell culture incubator and

both phase-contrast and fluorescent images were collected

every 2-3 hours.

Alterations in cell morphology were evident within only a few

hours of SSP treatment as illustrated in the phase image in

Figure 2A. Using fluorescent images, we positively identified

cells containing fluorescently stained DNA indicating

activation of caspase-3/7, cleavage of the DEVD moiety in the

kinetic apoptosis reagent, and fluorescent labeling of cellular

DNA (green image in Figure 2A). Using the object counting

algorithm, we successfully quantified the number of

fluorescent objects as indicated with red x’s in Figure 2A. The

object counting criteria were then applied to all images in the

experiment at each time point. The data in Figure 2B indicate

that caspase-3/7 activation is detectable within a few hours of

SSP treatment, with a maximal response triggered in the

presence of 333 nM SSP.

Increasing concentrations of SSP also significantly affected

cell proliferation. To demonstrate this on the IncuCyte FLR,

we completed an end point analysis at the 48 hour time point.

Vybrant DyeCycle Green DNA dye was added directly (no

wash required) to the wells at a final concentration of 1 µM in

50 µl of PBS. After a 30 minute incubation, the total number

of DNA containing objects was enumerated using the object

counting algorithm. As expected, our data indicate an inverse

correlation between the total number of objects and the

apoptotic index as a function of increasing concentrations of

SSP (Figure 2C). The data clearly indicate the advantage of

seeing all the kinetic time points as they occur, thereby

alleviating the need to pick an end-point for analysis a-priori

to running the experiment.

Multiplexed, kinetic measurements of proliferation and

apoptosis

The two fluorescent channels available on the IncuCyte

ZOOM provides a way to kinetically measure caspase-3/7

activation in addition to proliferation (nuclear label) within the

same well, thus eliminating the need for end-point analysis. In

the next experiment, HeLa NucLight Red cells were treated

with SSP in the presence of the 5µM caspase 3/7 reagent and

phase-contrast, red, and green images were collected every 2

hours in IncuCyte ZOOM using a 10x objective (Figure 3;

Supplemental Figure 1 - 4x data). These data illustrate typical

A

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CellPlayer™ 96-Well Kinetic Caspase-3/7 Apoptosis Assay

4

Figure 4: Extrinsic activation of caspase-3/7 in A549 lung epithelial cells. (A)

Caspase-3/7 activation of A549 lung epithelial cells in response to varying

concentrations of TNF-α in the presence of 5µg/ml cycloheximide (CHX). (B) The

EC50 value of TNF-α was calculated by using the area under the curve (AUC) of

caspase-3/7 objects/mm2 over time.

results obtained using the caspase 3/7 reagent multiplexed

with NucLight Red cells to measure the kinetic induction of

apoptosis and proliferative effects of drug treatment (Figure

3B and 3C, respectively). Using all of the kinetic data in

Figure 3B and 3C, area under the curve (AUC) values were

plotted and EC50 (apoptosis) and IC50 (proliferation) values

were calculated. This 2-color kinetic assay provides a

multiplex way to analyze the apoptotic and anti-proliferative

effects of various treatments.

Caspase-3/7 reagent kinetically measures extrinsic activation

of apoptosis

Depending on the cellular context, exposure of cells to

Transcription Necrosis Factor alpha (TNF-α) can induce either

pro-survival, or cell death pathways. When used in isolation,

TNF-α induces NFκB activity and subsequent expression of

pro-survival signaling molecules (e.g. FLIP, XIAP, A20).

Alternatively, when used in conjunction with cycloheximide

(CHX), itself an inhibitor of translation, TNF-α is a potent

inducer of apoptosis through caspase 3 mediated signaling

pathways. To demonstrate this using IncuCyte ZOOM, A549

epithelial carcinoma cells were treated with increasing

concentrations of TNF-α in the presence of 5µg/ml CHX.

Caspase-3/7 was activated in a TNF-α concentration

dependent manner (Figure 4A). The AUC values for caspase-

3/7 positive objects over time were then used to calculate the

EC50 value of 0.676 nM TNF-α (Figure 4B). Supplemental

data shows other examples of apoptosis assays tested on the

IncuCyte FLR and ZOOM, such as varying FBS

concentrations and the addition of taxol (Supplemental Figures

2 and 3). These data demonstrate the ability to test a variety of

apoptotic inducing conditions on many cell types using this

kinetic, multiplexed assay.

Using Images and Movies to Confirm Signaling

One of the major advantages of using the IncuCyte is the

ability to verify the quantified kinetic data with both phase

contrast and fluorescent images. Classical morphological

changes associated with apoptosis include: cell shrinkage,

membrane blebbing, nuclear condensation, and DNA

fragmentation. The time lapse sequence presented in Figure 5

highlights this advantage, illustrating the ability to use phase

contrast and fluorescent blended images to temporally

correlate the activation of caspase-3/7 and the loss of red

nuclei due to cell death with morphological changes in

response to treatment with SSP. Using the IncuCyte the

temporal responses in every well can be supplemented with

a “movie” of either a phase contrast, fluorescence or

blended time-lapse sequence. This ability significantly

enhances the confidence in the measured response and any

Figure 5: Time-lapse images and movies to detect SSP induced

apoptosis in HeLa cells. HeLa NucLight Red cells were treated with 300nM

SSP in the presence of 5µM Caspase-3/7 reagent and imaged in IncuCyte

ZOOM every 30 minutes. Time-lapse images and movies monitor changes in

morphology and confirm the activation of the green caspase-3/7 signal in

Figure 2B and the loss of the red nuclear signal in Figure 2C.

CellPlayer™ 96-Well Kinetic Caspase-3/7 Apoptosis Assay

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5 nM 3.41 0.64 Z' = 0.69

25 nM 9.38 2.35

75 nM 16.59 1.21

300 nM 27.66 2.30

Figure 6: Statistical reproducibility of apoptotic response of MDA-MB-231 cells to SSP. (A) 96-well platemap showing reproducibility of single-well responses to various concentrations of SSP on MDA-MB-231 cells. The CellPlayerTM Apoptosis Assay is amenable to single-shot screening. (B) Linear regression plot comparing two different test plates spiked with the same concentrations of SSP. (C) Statistical measures from the same two plates.

subsequent conclusions drawn from quantitative image

analysis. See Supplemental Figure 3 for time-lapse images

corresponding to data collected on an FLR.

Validation of the Essen CellPlayer Kinetic Apoptosis Assay

Multiple factors must be considered when choosing an assay.

These factors include, but are not limited to: 1) the statistical

reproducibility of the assay, 2) assay throughput, 3) the cost

including time, labor and reagents, 4) the added information

content, e.g. endpoint vs. kinetic, and 5) assay preparation

considerations, e.g. no-wash vs. multiple wash labeling. To

assess the statistical reproducibility of the assay, we completed

a series of experiments using multiple cell types and control

compounds. These experiments included measuring apoptosis

in both a breast cancer cell line (MDA-MB-231) as well as in

primary HUVECs.

Statistical Validation using MDA-MB-231 Cells

In the first series of experiments we sought to demonstrate the

statistical reproducibility of this assay using SSP induced

apoptosis in MDA-MB-231 cells. Individual wells of a 96-

well plate were spiked with three different concentrations of

SSP (5 nM, 25 nM, and 75 nM) in duplicate plates. Images

were taken at three hour intervals and the number of

fluorescent objects per unit area was plotted on a per-well

basis in a 96-well format as illustrated in Figure 6A. These

data clearly show that wells receiving low, moderate, and high

doses of an apoptosis inducing reagent are clearly discernible

from each other both visually and quantitatively. Using these data we completed a number of additional

statistical analyses. First, we evaluated the plate to plate

reproducibility of the assay by plotting the data from replicate

plates on different axes and analyzing the data using linear

regression. The resulting R2 value of 0.97 indicates a strong

correlation between identically treated wells on separate plates

indicating strong inter-plate reproducibility (Figure 6B).

Figure 6C represents several other assay parameters. As

indicated by the means and standard deviations of the

apoptotic index calculated for individual treatment groups,

strong intra-plate reproducibility was also observed. Both

plates had Z’ values exceeding 0.65[7]

Importantly, by strategically spiking both edge and interior

wells on the microplate with SSP, we were able to statistically

determine that well location did not alter the apoptotic

response i.e. we did not observe any “edge effects”. Together,

these data indicate that the kinetic apoptosis reagent can be

used in the IncuCyte FLR to generate statistically robust data

using a small number of replicate samples and is amenable to

medium throughput assays given the six-plate capacity of the

IncuCyte FLR platform.

We also evaluated the ability to calculate EC50 values from

each column of a 96-well plate. To do this, we treated each

row of MDA-MB-231 cells with 3-fold decreasing

concentrations of SSP, as illustrated in Figure 7. Again, we

observed highly reproducible kinetic inductions of caspase-3/7

activity correlating to decreasing concentrations of SSP. Using

the calculated apoptotic index, we also show how these data

CellPlayer™ 96-Well Kinetic Caspase-3/7 Apoptosis Assay

6

Figure 7: Reproducibility of single plate concentration response of MDA-MB-231 cells to SSP. (A) 96-well platemap showing the reproducibility of concentration response to high (top) and low (bottom) concentrations of SSP. (B) Statistical analysis of the concentration response from rows of plate described in A. (C) Consistency of 12 EC50 determinations from plate described in A.

can be used to generate EC50 values. The resulting data

revealed a very narrow range of calculated EC50 values with

an excellent Z-factor of 0.67. Since the IncuCyte FLR is

capable of holding six 96-well or 384-well assay plates, 576-

2304 wells (72-144, 8-pt concentration response) of kinetic

data can be obtained from one experimental trial.

Conclusions

This application note demonstrates the following attributes of

the Essen BioScience 96-Well CellPlayer Kinetic Caspase-3/7

Apoptosis assay.

1) The apoptotic signal relies on activation of Caspase-3/7, a

primary and irreversible “executioner” pathway in most

cell types (see Supplemental Figure 4 for specificity data).

2) Cells can be labeled with NucLight reagents to measure

cell proliferation and kinetically monitor anti-proliferative

effects of compounds in addition to activation of Caspase-

3/7

3) IC50 and EC50 values can be calculated using kinetic

area under the curve values of nuclear counts and

caspase-3/7 counts, respectively.

4) The assay format follows a homogeneous “mix and read”

protocol which can be run over multiple days in full

media. There are no wash or lifting steps required,

negating the concern that cells are lost during the

experiment or labeling process.

5) The assay provides a full kinetic readout of apoptotic

signaling over multiple days from within your cell culture

incubator. Aside from providing insight into the dynamics

and timing of the apoptotic signaling pathway, this

attribute eliminates the need for determining a single,

optimum, assay endpoint a-priori; something which can

vary considerably for different cell types and for different

compound treatment conditions.

6) The assay has high statistical reproducibility and can be

used both for single-point screening or concentration

response profiling. Given the six 96-well plate capacity

of the IncuCyte FLR and ZOOM, up to 576 wells, or 72,

8-pt response curves can be acquired on the same

experiment.

7) All data points and temporal data curves can be

subsequently validated by individual images or time-lapse

movies respectively. The kinetic readout of the IncuCyte

FLR and ZOOM provides both high contrast (HD) phase

as well as quantitative fluorescent imaging. These data

can be used to validate and confirm the integrated image

processing metrics provided in the IncuCyte FLR and

ZOOM analysis package.

Together, these attributes provide a new and unique assay for

apoptotic pathway analysis for both drug discovery as well as

basic cell biology research.

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12.3 nM 15.16 3.23

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1 11.96

2 24.78

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12 28.48

MEAN 24.42

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CellPlayer™ 96-Well Kinetic Caspase-3/7 Apoptosis Assay

7

About the IncuCyteTM Live-Cell Imaging System

The Essen BioScience IncuCyteTM Live-Cell Imaging System is a compact, automated microscope. The IncuCyteTM resides inside your standard tissue culture incubator and is used for long-term kinetic

imaging. To request more information about the IncuCyteTM, please visit us at www.essenbioscience.com.

References

1. Cotter TG: Apoptosis and Cancer: The Genesis of a

Research Field. Nat Rev Cancer 2009, 9(7):501-507.

2. Shi Y: Mechanisms of Caspase Activation and

Inhibition During Apoptosis. Mol Cell 2002,

9(3):459-470.

3. Thornberry NA, Rano TA, Peterson EP, Rasper DM,

Timkey T, Garcia-Calvo M, Houtzager VM,

Nordstrom PA, Roy S, Vaillancourt JP, Chapman

KT, Nicholson DW: A Combinatorial Approach

Defines Specificities of Members of the Caspase

Family and Granzyme B. Functional Relationships

Established for Key Mediators of Apoptosis. J Biol

Chem 1997, 272(29):17907-17911.

4. Daya S, Robets M, Isherwood B, Ingleston-Orme A,

Caie P, Teobald I, Eagle R, Carragher N: Integrating

an Automated in Vitro Combination Screening

Platform with Live-Cell and Endpoint Phenotypic

Assays to Support the Testing of Drug

Combinations. SBS 16th Annual Conference and

Exhibition 2010.

5. Cen H, Mao F, Aronchik I, Fuentes RJ, Firestone GL:

Devd-Nucview488: A Novel Class of Enzyme

Substrates for Real-Time Detection of Caspase-3

Activity in Live Cells. FASEB J 2008, 22(7):2243-

2252.

6. Janicke RU, Sprengart ML, Wati MR, Porter AG:

Caspase-3 Is Required for DNA Fragmentation and

Morphological Changes Associated with Apoptosis.

J Biol Chem 1998, 273(16):9357-9360.

7. Zhang JH, Chung TD, Oldenburg KR: A Simple

Statistical Parameter for Use in Evaluation and

Validation of High Throughput Screening Assays. J

Biomol Screen 1999, 4(2):67-73.

8000-0074-D00

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Supplemental Figure 2: Apoptotic response of HUVECs to serum

deprivation. (A) Kinetic measures of the number of caspase-3/7 positive

cells is recorded over time and plotted as fluorescent objects, n=3 wells per

data point shown (C) At the 62 hour end point, the apoptotic index was

calculated by dividing the number of caspase-3/7 fluorescent objects by the

total number of DNA containing objects following staining with Vybrant

DyeCycle Green.

Apoptosis data collected using a 4x objective in IncuCyte

ZOOM

The IncuCyte ZOOM allows users to easily change the

objective between 4x, 10x, and 20x. To confirm our data was

reproducible at different objectives, we treated HeLa

NucLight Red cells with Staurosporine (SSP) in the presence

of 5µM Caspase-3/7 and imaged with a 4x objective. EC50

(apoptosis) and IC50 (proliferation) data was comparable to

that experiments conducted using the 10x objective.

Measuring Apoptosis in Primary HUVECs

To determine the level of caspase-3/7 mediated apoptosis in

primary HUVECs in culture, low passage HUVECs were

plated in media with decreasing levels of serum

supplementation and 5 µM Caspase-3/7 apoptosis reagent,

placed in the IncuCyte FLR within an incubator, and data was

collected over the course of 3 days. As shown in Supplemental

Figure 2, positive identification of cells with activated

caspase-3/7 was observed within 20 hours of culture in cells

plated in media with less than 2% serum. Interestingly, we

also observed a significant amount of caspase-3/7 positive

cells in both media supplemented with 4% serum, and in

complete media arising at approximately 25-30 hours post

assay initiation. Using endpoint data, we found that nearly 5-

10% of HUVECs cultured in complete media were apoptotic.

These data illustrate another utility for this reagent,

Supplemental Figure 1: Caspase-3/7 and NucLight Red Data at 4x. (A)

Caspase-3/7 activation and (B) HeLa NucLight Red cell counts were

measured over time in response to SSP treatment using a 4x objective in

IncuCyte ZOOM. (C) Area under the curve data was then used to calculate

EC50 and IC50 values of SSP.

Vehicle

Control33 nM

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Supplemental Figure 3: Taxol induced caspase-3/7 mediated apoptosis in

human breast adenocarcinoma cells. MDA-MB-231 cells were treated with 33

nM taxol and the number of caspase-3/7 positive cells was quantified using the

IncuCyteTM object counting algorithm. The apoptotic index was calculated by

dividing the number of caspase-3/7 fluorescent objects by the total number of

DNA containing objects following staining with Vybrant DyeCycle Green. Bottom

panels, classical morphological changes associated with apoptotic cells were

observed using phase contrast images.

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MDA-MB-231 MCF-7 (CASP-3-/-)Vehicle Control

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Supplemental Figure 4: The Essen CellPlayerTM Kinetic Caspases-3/7 Apoptosis reagent requires caspase-3 for detection of apoptotic nuclei. SSP induced caspase-3/7 activity in MDA-MB-231 cells (CASP-3+/+) was compared to MCF-7 cells (CASP-3-/-). The number of fluorescent objects, correlative to caspase-3/7 activity, was determined using the IncuCyteTM object counting algorithm. The apoptotic index was calculated by dividing the number of caspase-3/7 fluorescent objects by the total number of DNA containing objects following staining with Vybrant DyeCycle Green.

specifically for use in cell culture quality control experiments.

Taxol induced Apoptosis

To illustrate another application and show time-lapse images

associated with activation of caspase-3/7, MDA-MB-231

breast adenocar-cinoma cells were treated with taxol in the

presence of Caspase-3/7 reagent.

The Essen CellPlayer Kinetic Caspase-3/7 Apoptosis reagent

specifically measures the activation of caspase-3/7

It is well known that addition of SSP can trigger apoptosis

through multiple pathways. Therefore, we sought to verify that

the kinetic apoptosis reagent was specific to caspase-3/7, and

not to other caspases. It has been demonstrated that the breast

carcinoma cell line, MCF-7, lacks caspase-3 expression[6].

This is a result of a 47-base pair deletion within exon 3 of the

CASP-3 gene resulting in altered mRNA splicing and the

introduction of a premature stop codon that halts translation of

the CASP-3 mRNA. Early studies showed that although MCF-

7 cells remain sensitive to both SSP and TNF-α induced

apoptosis, morphological alterations including shrinkage,

blebbing and DNA fragmentation were not observed

suggesting a critical role for caspase-3 in mediating these

apoptosis specific characteristics. Therefore, MCF-7 cells

were used to determine whether caspase-3 is required for

positive identification of apoptotic cells using this reagent

(Supplement Figure 4). To determine whether caspase-3 is

required for positive identification of apoptotic cells using this

reagent, we treated MCF-7 cells (CASP-/-) with a mid-range

concentration of 100 nM SSP in the presence of Caspase 3/7

reagent. We did not observe caspase-3/7 activation in MCF-7

cells at any time point tested (Supplemental Figure 4). In

contrast, a significant induction of caspase-3/7 activity was

detectable in identically treated MDA-MB-231 cells. Endpoint

calculations of percent apoptosis indicated a slight induction

of caspase-3/7 activation in 100 nM SSP treated MCF-7 cells.

It is possible that caspase-7, another executioner caspase that

cleaves at the DEVD motif, is responsible for this activity.

Nevertheless, these data clearly illustrate that the kinetic

apoptosis reagent can be utilized to measure caspase-3/7

activation induced by SSP, and that caspase-3 is required for

positive identification of apoptotic cells using this reagent.