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
0 4 12 37 111
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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
B
C
D
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
5
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[SSP] Apoptotic Index (Mean) SD
5 nM 3.53 0.38 Z' = 0.76
25 nM 9.32 1.39
75 nM 24.26 1.52
300 nM 41.40 3.04
Plate 2 - N = 6 per treatment
[SSP] Apoptotic Index (Mean) SD
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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1000 nM 60.50 6.05 Z' = 0.67
333 nM 60.33 5.62
111 nM 53.27 4.77
37 nM 32.79 3.10
12.3 nM 15.16 3.23
Column EC50 (nM)
1 11.96
2 24.78
3 34.51
4 36.63
5 16.22
6 18.71
7 22.45
8 31.34
9 21.02
10 29.22
11 30.73
12 28.48
MEAN 24.42
SD 6.74
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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4%
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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
Taxol
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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.
0 10 20 30 40 500
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iew
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MDA-MB-231 MCF-7 (CASP-3-/-)Vehicle Control
100 nM SSP
MDA-MB-231 MCF-7 (CASP-3-/-)
flu
ore
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/mm
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flu
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/mm
2
ap
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de
x
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.
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