Simplified evaluation of apoptosis using the MuseTM Cell Analyzer
ABSTRACT
Asima Khan
he degree of apoptosis in a cell population is an important parameter of cell health and
Tis characterized by distinct morphological changes. Current methods of accurate detec-
Katherine Gillis
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"!
Julie Clor
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.
Kamala Tyagarajan*
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
Millipore Corporation, 25801 Industrial Blvd,
cellular apoptosis. The assay is versatile and works with both suspension and adherent cell
Hayward, California, CA 94545, USA
lines and multiple treatment conditions.
*
Millipore Corporation, 25801 Industrial
INTRODUCTION
Blvd, Hayward, California, CA 94545, USA;
tel.: 510 576 1352, e-mail: kamala.tyagarajan@
The degree of apoptosis in a cell population is an important parameter that
merckgroup.com,
contributes to a comprehensive picture of cell health. Accurate detection and
measurement of cellular apoptosis is essential for drug development and discov-
Received: November 20, 2012
ery, for understanding mode of compound action, and for understanding the im-
Accepted: November 21, 2012
pact of culture and growth conditions. However, the assessment of cellular ap-
optosis has been limited due to the requirements for expensive and complicated
Keywords: apoptosis, cell death, Muse, cell
analysis, annexin, phosphatidylserine
instrument platforms, expertise and improved analytical methods that provide
rapid, robust and reproducible apoptosis data. Access to these improvements
Abbreviations: 7-AAD 7-Aminoactinomy-
can facilitate apoptosis monitoring, thereby enabling the efficient, daily execu-
cin D, CHO Chinese Hamster Ovary, PS
tion of cellular research.
phosphatidylserine
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.
492 www.postepybiochemii.pl
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 (+).
Figure 1. The combined use of fluorescent labeled Annexin V and membrane-
-impermeant 7-AAD DNA-binding dye can distinguish live, early apoptotic, and
SAMPLE PREPARATION
late apoptotic cells.
Chinese Hamster Ovary (CHO), HeLa, and Jurkat cells
The Muse"! Annexin V & Dead Cell Assay is based on were kept in log phase growth in complete medium. Prior
the detection of phosphatidylserine (PS) on the surface of to assaying, cells were treated with compounds as indi-
apoptotic cells and uses a premixed reagent containing flu- 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-
Figure 2. Workflow for Muse"! Annexin V & Dead Cell Assay. The assay utilizes a simple mix-and-read procedure and
scribed above. Key features of
provides quantitative apoptosis data.
the assay include:
orescently labeled Annexin V in combination with a dead mix-and-read assay minimizes loss of fragile, apoptotic
2+
cell marker, 7-AAD. Annexin V is a Ca -dependent phos- cells;
pholipid-binding protein that has a high affinity for PS, a highly simplified acquisition and analysis, guided
membrane component normally localized to the internal through touchscreen interface;
face of the cell membrane. Early in the apoptotic pathway, accurate and precise data;
molecules of PS are translocated to the outer surface of the minimal number of cells required;
cell membrane where Annexin V can readily bind to them validated with both suspension and adherent cell lines.
(Fig. 1). Late-stage apoptotic cells show loss of membrane
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.
Postępy Biochemii 58 (4) 2012 493
Figure 5. Superior precision for apoptotic percentage detection, compared to
other analysis methods. Data are based on triplicate measurements of 10 sam-
Figure 4. Apoptosis measurements are consistent with other cell analysis me- ples from suspension and adherent cell lines treated with staurosporine to induce
thods. CHO-K1 and Jurkat cells were treated with staurosporine to induce apop- apoptosis.
tosis. The data show the comparison of population percentages for all 4 methods
(Fluorescent Microscope, Image-Based Automated device, Personal cell Analyzer,
(Fig. 3). Briefly, a user enters the Annexin V & Dead Cell
and Muse"! cell Analyzer). Each point represents the average of three samplings.
Module and hits Run Assay . The touchscreen prompts
the user to load a sample and, through simple on-screen
DATA ACQUISITION
instructions, guides the user through the optimization
Data from prepared samples were quickly acquired us-
and verification of settings. The user then enters sample-
ing the step-by-step instructions on the touchscreen and
specific information and then touches Run Sample. The
the Muse"! Annexin V & Dead Cell Software Module
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
Figure 6. Apoptotic impacts of multiple compounds on Jurkat suspension cell line. untreated Jurkat
staurosporine for 4 h. Samples were stained
cells (Top, left) were compared with cells treated for 4 h with 10 µM camptothecin, a DNA topoiso-
in triplicate following the manufacturer s in-
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 structions and analyzed using either Muse"!
V & Dead Cell Assay.
Annexin V & Dead Cell Assay, fluorescent
494 www.postepybiochemii.pl
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
Figure 7. Analysis of apoptosis and cell death in HeLa cells induced by various compounds. Untre-
late apoptotic/dead cells.
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
Results from the analysis of the adherent
Muse"! Annexin V & Dead Cell Assay.
HeLa cell line are shown in Figure 7. Again,
the assay enabled clear discrimination of ap-
microscopy, image-based fluorescent analysis, or the
optotic and dead cell populations. Both ani-
guava® Personal Cell Analyzer (PCA). The results for the
somycin and diamide resulted in high percentages of late
percent of apoptotic and dead cells clearly indicate that
apoptotic or dead cells and very little early apoptosis,
the Muse"! Cell Analyzer provides equivalent cell pop-
while camptothecin treatment resulted in the appearance
ulation measurement results, compared to results from
of equal proportions of early and late apoptotic popula-
other predicate analysis methods, for both adherent and
tions.
suspension cell lines (Fig. 4).
Figure 9. Dose response data obtained for Jurkat cells treated with gambogic acid.
Figure 8. Dose response data obtained for Jurkat cells treated with staurosporine
Jurkat cells were treated with multiple concentrations of gambogic acid for 4 h
for 4 h. Jurkat cells were treated with multiple concentrations of staurosporine
then analyzed using the Muse"! Annexin V & Dead Cell Assay. Each data point
for 4 h then analyzed using the Muse"! Annexin V & Dead Cell Assay. Each data
represents a triplicate sampling at each concentration. Error bars represent stan-
point represents a triplicate sampling at each concentration.
dard deviation.
Postępy Biochemii 58 (4) 2012 495
DOSE RESPONSE STUDIES WITH MULTIPLE INDUCERS results indicate that the mix-and-read Muse"! Annexin V &
Dead Cell Assay and software module enabled the acqui-
Simplified dose response measurements are important
sition of accurate and highly precise measurements of cel-
to understanding mode of compound action. Jurkat cells
lular apoptosis. The assay is versatile and works with both
were treated with multiple concentrations of staurosporine,
suspension and adherent cell lines and multiple treatment
a known protein kinase inhibitor, for 4 h then analyzed sam-
conditions. The Muse"! Annexin V & Dead Cell Assay has
ples using the Muse"! Annexin V & Dead Cell Assay (Fig. 8).
the potential to greatly simplify the study of apoptosis and
At all concentrations, staurosporine treatment caused cells
enable researchers to easily obtain dose-based and mecha-
to primarily undergo early apoptosis, exhibiting little or no
nistic information in the drug discovery process for com-
death. On the other hand, gambogic acid, a compound with
pounds of interest.
potent anti-tumor activity, caused rapid apoptosis and cell
death at relatively low concentrations (Fig. 9). At concentra-
REFERENCES
tions of gambogic acid below 18.75 µM, a higher proportion
of early apoptotic cells were observed, while increasing con- 1. Gillis K, Clor J, Khan A, Tyagarajan K (2011) Precise and Accurate
Counts and Viability Measurements Across Multiple Cell Lines Using
centrations resulted in a predominance of late apoptotic/
the Muse"! Cell Count & Viability Assay. Merck Millipore Cellutions
dead cells, even given the short duration of treatment. These
Newsletter 4: 3-7
results are consistent with the established classification of
2. Guizzunti G, Batova A, Chantarasriwong O, Dakanali M, Theodora-
gambogic acid as a potent inducer of apoptosis [2].
kis EA (2012) Subcellular Localization and Activity of Gambogic Acid.
Chembiochem. 13: 1191-1198
CONCLUSIONS
The Muse"! Cell Analyzer provides a simple, powerful
method for obtaining important apoptosis information. Our
Uproszczona ocena procesu apoptozy z zastosowaniem
MuseTM 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
umierajÄ…ce w wyniku apoptozy wykazujÄ… 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.
496 www.postepybiochemii.pl
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