Early Detection of Apoptosis Using a
Fluorescent Conjugate of Annexin V
Guohong Zhang, Vanessa Gurtu, Steven R. Kain and Guochen Yan1
CLONTECH Laboratories, Palo Alto, CA and 1Sugen, Inc., Redwood City, CA, USA
BioTechniques 23:525-531 (September 1997)
ABSTRACT
Apoptosis of mammalian cells is accompanied by various morphological changes including nuclear condensation, DNA fragmentation and cell surface changes. Methods developed over the past
few years have focused on detection of DNA-associated changes that
occur rather late in apoptosis. However, detection of apoptosis at
early stages, before gross morphological changes, is critical for understanding the pathways of programmed cell death. In this report,
we describe a rapid and reliable assay for detecting early stages of
apoptosis. This assay is based on the observation that soon after initiating apoptosis, most mammalian cell types translocate phosphatidylserine (PS) from the inner face of the plasma membrane to
the cell surface. Once on the cell surface, PS can be specifically detected by staining with fluorescein isothiocyanate (FITC)-labeled
annexin V (annexin V-FITC), a protein with a strong, natural affinity
for PS. Using this assay, we have detected apoptotic cells in culture,
in real time, using fluorescence microscopy and flow cytometry. In
combination with vital dye staining, the progressive stages of apoptosis were observed. PS redistribution occurs earlier than DNA-associated changes and membrane leakage. In addition, PS externalization occurs during apoptosis induced by a variety of stimuli.
Therefore, the annexin V binding assay provides an excellent indicator for the early stages of apoptosis.
INTRODUCTION
Apoptosis is a fundamental feature of many biological
processes. This mode of cell death culminates in early recognition (i.e., before plasma membrane rupture) of dying cells
by phagocytes and appears to have been highly conserved
Vol. 23, No. 3 (1997)
throughout evolution (5,15,16,23,24). Apoptosis can be triggered by a diverse array of stimuli, and is characterized by a
number of morphological changes. These include cell shrinkage, chromatin condensation, DNA fragmentation, membrane
blebbing and formation of apoptotic bodies (4,12,32).
A number of methods exist for detecting apoptotic cells.
Loss of cell viability (failure to either exclude vital dyes or
transform tetrazolium salts to colored products), DNA fragmentation (assayed by agarose gel electrophoresis or in situ
terminal transferase labeling) and DNA condensation (detected by Hoechst dye staining of nuclear DNA) are some of the
traits used to monitor apoptosis. Among these, the methods
that provide quantitative data lack specificity, are time-consuming and usually require the destruction of cell integrity
(6,17,19,22,25,27,28,31). In addition, the morphological
changes and degradation of chromatin, which are the basis of
such assays, occur rather late in apoptosis (13,30).
An early and critical event in apoptosis involves the acquisition of surface changes by the dying cells that results in the
recognition and uptake of these apoptotic cells by phagocytes
(5,11,13,15,16,24). However, changes on the cell surface of
apoptotic cells, such as the expression of thrombospondin
binding sites (20), loss of sialic acid residues (23) and exposure of the membrane phospholipid, phosphatidylserine (PS;
References 8 and 9) have been difficult to detect.
Annexin V is a calcium-dependent, phospholipid-binding
protein that preferentially binds PS (1,26,29). Recent studies
have demonstrated that human neutrophils lose their surface
FcγRIII and acquire annexin V binding sites during apoptosis
in vitro (10). The cell surface component that is specifically
bound by annexin V was demonstrated to be PS (1,26,29). PS
is normally confined to the inner (cytoplasmic) face of the
BioTechniques 525
plasma membrane (18), but translocates to the cell surface in
apoptotic cells (7,8,29). Fadok et al. have reported that PS externalization mediates macrophage recognition of apoptotic
cells (9). Martin and coworkers have further demonstrated
that the PS redistribution is induced by a variety of apoptotic
stimuli and occurs in a wide variety of cell types (13).
The specificity of annexin V binding to PS has been
demonstrated through inhibition studies using fluorescein
isothiocyanate (FITC)-labeled annexin V (annexin V-FITC)
as a probe (13). Binding of annexin V to cell surface PS was
inhibited in the presence of PS liposomes, but was unaffected
by liposomes containing other phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and sphingomyelin (13). Annexin V-FITC has also
been used successfully to detect PS exposure during platelet
activation, a major source of procoagulant activity (21,29);
serum withdrawal-induced apoptosis of marine germinal center B cells (11); and anti-Fas antibody-induced apoptosis in
Jurkat cells (13).
In this report, we describe a one-step assay for apoptosis
using annexin V-FITC. This assay detects the early stages of
apoptosis, before detection of chromosomal-based changes.
We show that PS externalization precedes the changes in
membrane permeability and nuclear condensation, and occurs
in a stimulus-independent manner.
Annexin V-FITC Binding Assay
Apoptosis was induced by various stimuli as indicated. Annexin V binding assays were performed using ApoAlert Annexin V Apoptosis Kit (CLONTECH Laboratories, Palo Alto,
CA, USA). Apoptotic cells were identified either by direct visualization of green-colored membrane staining under a fluorescence microscope or by flow cytometry. To distinguish
cells that had lost membrane integrity, propidium iodide (PI)
was added to a final concentration of 10 µg/mL before analysis. Hoechst dye staining was performed to reveal nuclear condensation and was added at a final concentration of 1 µg/mL.
Scoring of Apoptosis
Apoptotic cells were scored under a Zeiss Axioskop Fluorescence Microscope (Carl Zeiss, Thornwood, NY, USA).
Generally, 4–6 representative fields of at least 100 cells were
scored for annexin V-FITC binding as shown by green-colored membrane staining. The same population of cells was
counted for staining with Hoechst 33342 dye as demonstrated
by bright, blue-colored nuclear staining. Flow cytometric
analysis was performed as previously described (13).
RESULTS
MATERIALS AND METHODS
Visualization of Progressive Stages of Apoptosis
Cell Culture and Supplements
Previously, it has been reported that fluorescein-labeled
annexin V can detect PS exposure on apoptotic cells by flow
cytometry (13,30). To evaluate whether annexin V binding to
PS on the cell surface can be an effective means of quantifying apoptosis, we established the annexin V-FITC staining
procedure. Figure 1 illustrates the biological basis of the
annexin V-FITC staining assay. Using the assay, we have
32D cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), and 5 ng/mL
mouse Interleukin 3 (IL-3), 100 U/mL of penicillin and 100
µg/mL of streptomycin (all from Sigma Chemical, St. Louis,
MO, USA).
Figure 1. The biological basis of the annexin V-FITC binding assay. In normal cells, PS is predominantly located on the inner leaflet of the plasma membrane. When cells initiate apoptosis, PS is rapidly translocated to the outer leaflet. In the presence of Ca2+, annexin V binds PS with high affinity.
526 BioTechniques
Vol. 23, No. 3 (1997)
Table 1. Time-Dependent Induction of Apoptosis as Analyzed by Annexin V-FITC and Hoechst 33342 Staining
Table 2. Induction of Apoptosis in 32D Cells by a Variety of Apoptotic
Stimuli
% Annexin V
Positive Cells
% Hoechst 33342
Positive Cells
Hours
% Annexin V
Positive Cells
% Hoechst 33342
Positive Cells
Stimuli
0
6.6 ± 9.4
6.6 ± 9.0
Control
10.2 ±
2.3
6.5 ± 2.2
6
11.3 ± 5.7
3.0 ± 1.4
Actinomycin D
48.0 ± 27.5
2.5 ± 1.1
8
22.6 ± 11.7
3.3 ± 2.3
Cycloheximide
48.2 ±
9.4
23.0 ± 4.6
15
34.3 ± 7.5
10.0 ± 2.9
Staurosporine
17.0 ±
5.4
7.2 ± 5.1
24
40.0 ± 14.1
26.0 ± 5.3
Etoposide/VP-16
58.0 ± 10.7
49.2 ± 24.4
UV Irradiation
44.5 ± 12.8
9.2 ± 6.2
Apoptosis was induced in 32D cells by removal of IL-3
from the culture medium for various time intervals as indicated. Annexin V-FITC and Hoechst dye staining were
performed as described in Materials and Methods. The
percentages of annexin V and Hoechst dye positive cells
were determined by scoring the cells positive for greencolored membrane staining with annexin V-FITC and bluecolored condensed nuclear staining with Hoechst dye, respectively. Generally, 4–6 representative fields of at least
100 cells were scored.
visualized apoptotic cells in real time with live cells using fluorescence microscopy. For comparison studies, we have also
used Hoechst dye and PI staining, which detect nuclear condensation and membrane leakage, respectively.
Apoptosis was induced in 32D cells by incubating the cells
with 5% ethanol (13) for various time intervals as indicated.
Figure 2 shows the progressive stages of apoptosis as monitored by annexin V binding, in conjunction with Hoechst dye
and PI staining. The cell in Panel A shows early apoptotic
staining (green) with annexin V-FITC, but does not show any
of the yellow-orange PI fluorescence seen in another apoptotic cell in the late stage as shown in Panel D. Similarly, the
mid-stage cell in Panel B does not stain with PI; however,
Hoechst dye staining (blue) of the same cell reveals the onset
of nuclear condensation (Panel C). The strong, yellow signal
in Panel D is due to the free movement of PI across the plasma membrane. Increased membrane permeability is characteristic of cells in the later stages of apoptosis. Therefore, PS
exposure as detected by annexin V-FITC binding appears to
be an early event during apoptosis. Our data further suggest
that, in combination with vital dye staining, the annexin VFITC binding assays can be used to monitor the progressive
stages of apoptosis.
Apoptosis was induced in 32D cells for 4 h by actinomycin
D (50 µM), cycloheximide (100 µM), staurosporine (1 µM)
and etoposide (100 µM). For induction by UV, cells were irradiated under UV for 5 min followed by incubation for 4 h.
Annexin V-FITC and Hoechst dye assays were performed
as described in the Materials and Methods. The percentage of annexin V-FITC and Hoechst dye-positive cells
were scored as described in the legend to Table 1.
nexin V-FITC-stained cells were detected at 6 h, whereas
cells stained positive for Hoechst 33342 were not detected until 15 h after induction (Table 1). In addition, at each time
point, the percentage of cells exhibiting externalized PS was
significantly higher than that showing DNA condensation
(Table 1). These data suggest that PS externalization occurs
more rapidly and is more prevalent than nuclear changes
PS Exposure on Apoptotic Cells Occurs Earlier than
Nuclear Changes
To further evaluate whether annexin V binding of PS can
be used as a reliable marker for detecting early stages of
apoptosis, we compared time-course studies of PS externalization to nuclear DNA condensation. Apoptosis was induced
in 32D cells (3) for various time intervals as indicated. PS externalization and nuclear condensation were assessed at each
time point by counting the cells positive for annexin V-FITC
staining on cell membrane, and Hoechst dye staining for condensed nuclear DNA, under a fluorescence microscope. An528 BioTechniques
Figure 2. Progressive stages of apoptosis monitored by annexin V-FITC
binding assay. Apoptosis was induced in 32D cells by incubation with 5%
ethanol for 60 min (A), 90 min (B and C) and 120 min (D). Annexin V binding assay, Hoechst 33342 and PI staining were performed as described in Materials and Methods. Cells were photographed with a Zeiss fluorescence microscope (A and D: dual-pass FITC/rhodamine filter set; B and C: FITC and
4′,6-diamidino-2-phenylindole [DAPI] filter sets, respectively).
Vol. 23, No. 3 (1997)
associated with apoptosis. Therefore, the annexin V binding
assay detects apoptotic cells significantly earlier than detections based on alteration in gross DNA structures.
PS Externalization Occurs in a Stimulus-Independent
Manner
Apoptosis occurs in many different systems and in response to many different external stimuli. Thus, the ideal
apoptotic marker should give a consistent signal regardless of
the apoptotic stimulus. Many types of reagents have been
shown to induce apoptosis through different signaling events
that lead to the common biochemical and morphological
changes associated with apoptosis (2,4,14). To determine
whether PS externalization accompanies apoptosis under different inducing conditions, we analyzed annexin V binding of
32D cells following treatment of the cells with various apoptosis stimuli. All apoptosis-inducing conditions tested led to
an increase in annexin V binding of 32D cells as analyzed by
fluorescence microscopy (Table 2). Similarly, annexin VFITC also detected apoptosis in Jurkat cells treated with these
same reagents as analyzed by flow cytometry (Figure 3). In
addition, the annexin V-FITC assay consistently detected a
higher percentage of apoptotic cells than did assays based on
DNA condensation under all apoptosis-inducing conditions
tested (Table 2), consistent with the results described in Table
1. Taken together, these results indicate that PS externalization is a stimulus-independent event occurring at the early
stages of apoptosis.
DISCUSSION
We have shown that annexin V can be used in a simple,
non-invasive assay for early detection of apoptosis. In normal
cells, PS is located on the inner surface of the plasma membrane. Induction of apoptosis results in translocation of PS
from the inner to the outer surface of the plasma membrane,
apparently through an active mechanism (e.g., translocase;
Reference 13). Using the annexin V-FITC as a probe, we have
provided direct evidence that PS exposure is a widespread
event during apoptosis that occurs earlier than DNA-associated changes and membrane leakage. Therefore, annexin V
binding provides a useful general assay for detecting the onset
of cell death.
Chromatin fragmentation assays based on DNA separation
(DNA laddering) and in situ detection are conventional methods for detecting apoptosis. However, these methods involve
many steps, are very time-consuming and cannot be used with
living cells. In addition, these methods detect only the later
stages of apoptosis (Tables 1 and 2; Reference 13). Annexin
V binding assays have several advantages over these existing
methods; for example, annexin V binding requires only 5–10
min, whereas the DNA-fragmentation-based assays take 3–4
h to complete. In addition, annexin V binding is nonenzymatic and does not require fixation, so it allows one to score
apoptotic cells with living, unfixed samples, which is not possible with conventional apoptosis assays. Furthermore, annexin V binding detects early stages of apoptosis and thus
provides an early marker for downstream study of apoptotic
pathways. However, since annexin V positive cells may also
be necrotic, PI staining is recommended to accompany the annexin V procedure.
Vol. 23, No. 3 (1997)
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Figure 3. Flow cytometric analysis of apoptosis induced by various stimuli. Apoptosis was induced in Jurkat cells by various stimuli for 4 h or 5 min under
UV irradiation followed by 4-h incubation. Flow cytometric analysis of annexin V binding was performed as described in Materials and Methods.
Apoptosis has become an important biological phenomenon for researchers studying cancer, development, DNA damage and gene repair. The ability to detect the early stages of
apoptosis with living, unfixed cells using annexin V binding
allows many experimental options that are incompatible with
conventional apoptosis assays. For example, it should be possible to use the annexin V binding to select early apoptotic
cells from a population by fluorescence-activated cell sorting
(FACS) and then monitor the progress of these cells through
the late stages of apoptosis. Thus, annexin V binding provides
an early marker for studying downstream apoptotic pathways.
In addition, it should be possible using the annexin V binding
assay to quantify the kinetics of progressive cell death over
time and in relation to the cell cycle. Further studies are required to evaluate whether PS translocation may also be involved in other cellular processes.
Note added in proof: Following submission of this manuscript, we have developed additional derivatives of annexin V,
including Cy3, biotin and GFP conjugates.
ACKNOWLEDGMENTS
We express our appreciation to Dr. Seamus Martin for
helpful information and technical assistance. We thank T.J.
Provost and D. Gunn for preparation of the Figures. We gratefully acknowledge Drs. P. Diehl, P. Seibert, J. Ambroziak and
D. Gunn for careful reading of the manuscript and useful discussion.
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Address correspondence to Guohong Zhang, CLONTECH
Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA
94303, USA. Internet: egzhang@clontech.com
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