492
www.postepybiochemii.pl
Asima Khan
Katherine Gillis
Julie Clor
Kamala Tyagarajan
*
Millipore Corporation, 25801 Industrial Blvd,
Hayward, California, CA 94545, USA
*
Millipore Corporation, 25801 Industrial
Blvd, Hayward, California, CA 94545, USA;
tel.: 510 576 1352, e-mail: kamala.tyagarajan@
merckgroup.com,
Received: November 20, 2012
Accepted: November 21, 2012
Keywords: apoptosis, cell death, Muse, cell
analysis, annexin, phosphatidylserine
Abbreviations: 7-AAD — 7-Aminoactinomy-
cin D, CHO — Chinese Hamster Ovary, PS —
phosphatidylserine
Simplified evaluation of apoptosis using the Muse
TM
Cell Analyzer
AbsTrACT
T
he degree of apoptosis in a cell population is an important parameter of cell health and
is characterized by distinct morphological changes. Current methods of accurate detec-
tion and measurement of cellular apoptosis require expensive and complicated instrument
platforms and expertise. The Muse™ Cell Analyzer is a unique instrument that enables mul-
tidimensional cell health analysis on a single platform. In this study, we used the Muse™
Cell Analyzer for apoptosis studies using the Muse™ Annexin V & Dead Cell Assay. The
assay is based on the detection of phosphatidylserine (PS) on the surface of apoptotic cells.
The results obtained from Muse™ Cell Analyzer were compared with traditional methods
for apoptosis analysis. Our results indicate that Muse™ Annexin V & Dead Cell Assay and
software module enabled the acquisition of accurate and highly precise measurements of
cellular apoptosis. The assay is versatile and works with both suspension and adherent cell
lines and multiple treatment conditions.
INTRODUCTION
The degree of apoptosis in a cell population is an important parameter that
contributes to a comprehensive picture of cell health. Accurate detection and
measurement of cellular apoptosis is essential for drug development and discov-
ery, for understanding mode of compound action, and for understanding the im-
pact of culture and growth conditions. However, the assessment of cellular ap-
optosis has been limited due to the requirements for expensive and complicated
instrument platforms, expertise and improved analytical methods that provide
rapid, robust and reproducible apoptosis data. Access to these improvements
can facilitate apoptosis monitoring, thereby enabling the efficient, daily execu-
tion of cellular research.
The Muse™ Cell Analyzer is a novel instrument that enables multidimen-
sional cell health analysis on a single platform. This small, benchtop cell analyzer
effortlessly guides users through the acquisition and analysis of samples using a
highly simplified and intuitive touchscreen interface which delivers rapid meas-
urements of cell concentration, viability, apoptotic status, and cell cycle distri-
bution [1]. Using multiparametric fluorescent detection of individual cells via
microcapillary flow technology, the system enables highly sensitive and rapid
detection of cellular samples using minimal cell numbers. The simplified format
enables researchers of varying backgrounds and experience levels to obtain a
comprehensive picture of cellular health.
In this study, we used the Muse™ Cell Analyzer for apoptosis studies using
the Muse™ Annexin V & Dead Cell Assay, a rapid, no-wash assay for the iden-
tification of apoptotic cells. Jurkat and HeLa cells were treated with a range of
cytotoxic and anti-tumor compounds, and the results from percentage of live,
early, late apoptotic, and dead cells were evaluated. Our results demonstrate
that the assay provides rapid and sensitive detection of cellular apoptosis and
provides quantitative and precise results on a variety of cellular types and com-
pound treatments.
MATERIALS AND METHODS
ASSAY PRINCIPLE
Apoptosis, or programmed cell death, is an important regulator of cell
growth and proliferation and is characterized by distinct morphological
changes. One key early aspect of apoptosis is the translocation of phosphati-
dylserine from the inner to the outer leaflet of the plasma membrane and
exposure to outer surface of the cell. This universal phenomenon is inde-
pendent of species, cell type and induction system and occurs early in the
apoptotic process.
Postępy Biochemii 58 (4) 2012
493
The Muse™ Annexin V & Dead Cell Assay is based on
the detection of phosphatidylserine (PS) on the surface of
apoptotic cells and uses a premixed reagent containing flu-
orescently labeled Annexin V in combination with a dead
cell marker, 7-AAD. Annexin V is a Ca
2+
-dependent phos-
pholipid-binding protein that has a high affinity for PS, a
membrane component normally localized to the internal
face of the cell membrane. Early in the apoptotic pathway,
molecules of PS are translocated to the outer surface of the
cell membrane where Annexin V can readily bind to them
(Fig. 1). Late-stage apoptotic cells show loss of membrane
integrity. The membrane-impermeant dye, 7-AAD, is used
to distinguish dead cells from early apoptotic cells. The as-
say can thus distinguish four populations:
— viable cells, not undergoing detectable apoptosis: An-
nexin V (–) and dead cell marker (–);
— early apoptotic and dead cells: Annexin V (+) and dead
cell marker (–);
— late apoptotic or dead cells: Annexin V (+) and dead cell
marker (+);
— cells that have died through non-apoptotic pathway: An-
nexin V (–) and dead cell marker (+).
SAMPLE PREPARATION
Chinese Hamster Ovary (CHO), HeLa, and Jurkat cells
were kept in log phase growth in complete medium. Prior
to assaying, cells were treated with compounds as indi-
cated in Figures 4–9. The assay employs a simple mix-
and-read procedure (Fig. 2).
100 µl of cells was mixed with
100 µl of Muse™ Annexin V &
Dead Cell Assay Reagent in 1.5
ml screw-cap microfuge tubes
and incubated for 20 min in
the dark at room temperature.
The assay provides results
on counts and concentrations
of the four cell populations de-
scribed above. Key features of
the assay include:
— mix-and-read assay minimizes loss of fragile, apoptotic
cells;
— highly simplified acquisition and analysis, guided
through touchscreen interface;
— accurate and precise data;
— minimal number of cells required;
— validated with both suspension and adherent cell lines.
Figure 1. The combined use of fluorescent labeled Annexin V and membrane-
-impermeant 7-AAD DNA-binding dye can distinguish live, early apoptotic, and
late apoptotic cells.
Figure 2. Workflow for Muse™ Annexin V & Dead Cell Assay. The assay utilizes a simple mix-and-read procedure and
provides quantitative apoptosis data.
Figure 3. Steps to perform acquisition and analysis using a guided user interface and software module. Results on apoptotic cell percentages and concentrations are di-
splayed automatically at the completion of acquisition. Optional dotplots allow for visualization and further data manipulation.
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DATA ACQUISITION
Data from prepared samples were quickly acquired us-
ing the step-by-step instructions on the touchscreen and
the Muse™ Annexin V & Dead Cell Software Module
(Fig. 3). Briefly, a user enters the Annexin V & Dead Cell
Module and hits “Run Assay”. The touchscreen prompts
the user to load a sample and, through simple on-screen
instructions, guides the user through the optimization
and verification of settings. The user then enters sample-
specific information and then touches “Run Sample.” The
instrument displays the results screen with the calculated
concentrations of live, early apoptotic, late
apoptotic, and dead cells. The instrument dis-
plays the results screen with the values and
provides the user the option to view the dot-
plot as well as adjust markers between sam-
ples. Result parameters include information
on:
— population percentages and concentra-
tions;
— total cells per ml;
— dilution factor (input value);
— sample number;
— sample ID.
Data can be stored on the device, exported
in a report format and/or exported as a Micro-
soft Excel
®
file, enabling the production of a
robust documentation trail with experimental
details preserved.
RESULTS
COMPARISON OF ACCURACY
WITH ALTERNATE METHODS OF
APOPTOSIS MEASUREMENT
The accuracy of test results obtained on the
Muse™ Cell Analyzer was compared with
traditional methods for apoptosis analysis.
Adherent CHO-K1 cells were treated with
0.25 µM staurosporine for 16 h and suspen-
sion Jurkat cells were treated with 1 µM
staurosporine for 4 h. Samples were stained
in triplicate following the manufacturer’s in-
structions and analyzed using either Muse™
Annexin V & Dead Cell Assay, fluorescent
Figure 4. Apoptosis measurements are consistent with other cell analysis me-
thods. CHO-K1 and Jurkat cells were treated with staurosporine to induce apop-
tosis. The data show the comparison of population percentages for all 4 methods
(Fluorescent Microscope, Image-Based Automated device, Personal cell Analyzer,
and Muse™ cell Analyzer). Each point represents the average of three samplings.
Figure 5. Superior precision for apoptotic percentage detection, compared to
other analysis methods. Data are based on triplicate measurements of 10 sam-
ples from suspension and adherent cell lines treated with staurosporine to induce
apoptosis.
Figure 6. Apoptotic impacts of multiple compounds on Jurkat suspension cell line. untreated Jurkat
cells (Top, left) were compared with cells treated for 4 h with 10 µM camptothecin, a DNA topoiso-
merase inhibitor (Top, right), 4.7 µM gambogic acid, a compound with potent anti -tumor activity
(Bottom, left), or 150 µM diamide a thiol-oxidizing agent (Bottom, right) using the Muse™ Annexin
V & Dead Cell Assay.
Postępy Biochemii 58 (4) 2012
495
microscopy, image-based fluorescent analysis, or the
guava
®
Personal Cell Analyzer (PCA). The results for the
percent of apoptotic and dead cells clearly indicate that
the Muse™ Cell Analyzer provides equivalent cell pop-
ulation measurement results, compared to results from
other predicate analysis methods, for both adherent and
suspension cell lines (Fig. 4).
EVALUATION OF PRECISION
AND REPRODUCIBILITY
To assess precision, triplicates of 10 sam-
ples from adherent and suspension cell lines
treated with staurosporine to induce apopto-
sis were prepared and analyzed them using
Annexin V-based assays using the fluorescent
microscope, an image-based fluorescent de-
vice, or the Muse™ Cell Analyzer. Average
coefficients of variation (%CVs) for the early
and late apoptotic populations were calculat-
ed for all devices and compared. The average
%CVs obtained with the Muse™ Annexin V &
Dead Cell Assay were consistently lower than
those seen using other comparative methods
for the same samples (Fig. 5).
STUDYING APOPTOTIC IMPACTS OF
COMPOUNDS ON MULTIPLE CELL TYPES
The apoptotic effects of several compounds
were evaluated using Jurkat cells (suspen-
sion) and HeLa cells (adherent). The results in
Figure 6 demonstrate that the differential im-
pacts on Jurkat cell health caused by the com-
pounds could be discriminated by the assay.
Camptothecin treatment caused early apop-
tosis with relatively little cell death, gambog-
ic acid treatment caused early apoptosis as
well as low levels of cell death, and diamide
resulted in the appearance of predominantly
late apoptotic/dead cells.
Results from the analysis of the adherent
HeLa cell line are shown in Figure 7. Again,
the assay enabled clear discrimination of ap-
optotic and dead cell populations. Both ani-
somycin and diamide resulted in high percentages of late
apoptotic or dead cells and very little early apoptosis,
while camptothecin treatment resulted in the appearance
of equal proportions of early and late apoptotic popula-
tions.
Figure 8. Dose response data obtained for Jurkat cells treated with staurosporine
for 4 h. Jurkat cells were treated with multiple concentrations of staurosporine
for 4 h then analyzed using the Muse™ Annexin V & Dead Cell Assay. Each data
point represents a triplicate sampling at each concentration.
Figure 7. Analysis of apoptosis and cell death in HeLa cells induced by various compounds. Untre-
ated HeLa cells (Top, left) were compared with cells treated for 16 h with 100 µM anisomycin, an
inhibitor of DNA synthesis (Top, right), 20 µM camptothecin, an inhibitor of the DNA enzyme to-
poisomerase I (Bottom, left), or 150 µM diamide, a thiol-oxidizing agent (Bottom, right) using the
Muse™ Annexin V & Dead Cell Assay.
Figure 9. Dose response data obtained for Jurkat cells treated with gambogic acid.
Jurkat cells were treated with multiple concentrations of gambogic acid for 4 h
then analyzed using the Muse™ Annexin V & Dead Cell Assay. Each data point
represents a triplicate sampling at each concentration. Error bars represent stan-
dard deviation.
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www.postepybiochemii.pl
Uproszczona ocena procesu apoptozy z zastosowaniem
Muse
TM
analizatora komórek
Asima Khan, Katherine Gillis, Julie Clor, Kamala Tyagarajan
*
Millipore Corporation, 25801 Industrial Blvd, Hayward, California, CA 94545, USA
*
e-mail: kamala.tyagarajan@merckgroup.com,
Słowa kluczowe: apoptoza, śmierć komórki, Muse, analiza komórek, aneksyna, fosfatydyloseryna
STRESzCzENIE
Stopień procesu apoptozy w populacji komórek jest ważnym czynnikiem pozwalającym na określenie parametrów ich zdrowia. Komórki
umierające w wyniku apoptozy wykazują szereg charakterystycznych, morfologicznych zmian. Obecne metody detekcji i pomiaru procesu
apoptozy wymagają kosztownych i skomplikowanych urządzeń oraz wiedzy eksperckiej do ich obsługi. Muse™ analizator komórek jest uni-
kalnym urządzeniem, które umożliwia wieloparametryczną analizę komórek. W artykule zaprezentowano zastosowanie Muse™ analizatora
komórek do badania procesu apoptozy, z wykorzystaniem zestawu „Muse™ Annexin V & Dead Cell Assay”. zasada działania testu oparta
jest na detekcji fosfatydyloseryny (PS) na powierzchni komórek apoptotycznych. Wyniki otrzymane z zastosowaniem Muse™ analizatora
komórek były porównywane z tradycyjnymi metodami używanymi do analizy procesu apoptozy. Otrzymane rezultaty wskazują, że zastoso-
wany zestaw do badań „Muse™ Annexin V & Dead Cell Assay” oraz dedykowane oprogramowanie do jego obsługi umożliwiają otrzymanie
dokładnych i precyzyjnych danych pomiarowych na temat procesu apoptozy komórek. Używany zestaw jest uniwersalny i można go stoso-
wać do badania zawiesinowych i adherentnych linii komórkowych, w różnych warunkach doświadczalnych.
DOSE RESPONSE STUDIES WITH MULTIPLE INDUCERS
Simplified dose response measurements are important
to understanding mode of compound action. Jurkat cells
were treated with multiple concentrations of staurosporine,
a known protein kinase inhibitor, for 4 h then analyzed sam-
ples using the Muse™ Annexin V & Dead Cell Assay (Fig. 8).
At all concentrations, staurosporine treatment caused cells
to primarily undergo early apoptosis, exhibiting little or no
death. On the other hand, gambogic acid, a compound with
potent anti-tumor activity, caused rapid apoptosis and cell
death at relatively low concentrations (Fig. 9). At concentra-
tions of gambogic acid below 18.75 µM, a higher proportion
of early apoptotic cells were observed, while increasing con-
centrations resulted in a predominance of late apoptotic/
dead cells, even given the short duration of treatment. These
results are consistent with the established classification of
gambogic acid as a potent inducer of apoptosis [2].
CONCLUSIONS
The Muse™ Cell Analyzer provides a simple, powerful
method for obtaining important apoptosis information. Our
results indicate that the mix-and-read Muse™ Annexin V &
Dead Cell Assay and software module enabled the acqui-
sition of accurate and highly precise measurements of cel-
lular apoptosis. The assay is versatile and works with both
suspension and adherent cell lines and multiple treatment
conditions. The Muse™ Annexin V & Dead Cell Assay has
the potential to greatly simplify the study of apoptosis and
enable researchers to easily obtain dose-based and mecha-
nistic information in the drug discovery process for com-
pounds of interest.
REFERENCES
1. Gillis K, Clor J, Khan A, Tyagarajan K (2011) Precise and Accurate
Counts and Viability Measurements Across Multiple Cell Lines Using
the Muse™ Cell Count & Viability Assay. Merck Millipore Cellutions
Newsletter 4: 3-7
2. Guizzunti G, Batova A, Chantarasriwong O, Dakanali M, Theodora-
kis EA (2012) Subcellular Localization and Activity of Gambogic Acid.
Chembiochem. 13: 1191-1198