plik

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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

Cell Analyzer

AbsTrACT

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.

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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

-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.

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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

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