Cell Biology International 30 (2006) 924e932

www.elsevier.com/locate/cellbi

Analysis of cycloheximide-induced apoptosis in human

leukocytes: Fluorescence microscopy using annexin V/propidium
iodide versus acridin orange/ethidium bromide
Dejan Baskic a,*, Suzana Popovic a, Petar Ristic b, Nebojsa N. Arsenijevic a
a

Institute of Microbiology and Immunology, School of Medicine, University of Kragujevac, Serbia and Montenegro
b
Public Health Institute, Kragujevac, Serbia and Montenegro
Received 30 January 2006; revised 3 June 2006; accepted 26 June 2006

Abstract
Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many
pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death.
In this study, we compared several methods for detecting apoptosis and necrosis in human leukocytes. Apoptosis was induced either by
incubating the cells with various doses of cycloheximide (CHX) or by 312 nm UVB irradiation. The methods used for detecting apoptosis
were light microscopy (May GrunwaldeGiemsa and trypan blue staining), fluorescence microscopy (acridin orange/ethidium bromide and
annexin V/propidium iodide staining) and agarose gel electrophoresis of fragmented genomic DNA.
Our study showed that CHX-induced apoptosis in cultured peripheral blood mononuclear cells but had no effect on apoptosis in polymorphonuclear cells, so its effect depends on cell type. Evaluation and comparison of the methods for detecting apoptosis showed the following. A
Giemsa-stained cytospin allows the main morphological characteristics of necrotic and apoptotic death to be recognized. Trypan blue staining,
widely used for estimating cell viability, is valueless for detecting apoptosis. Both fluorescence methods provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells, and were closely intercorrelated. Although applicable to a wide spectrum of cell types, agar electrophoresis of extracted DNA cannot be applied to all cell types and apoptotic conditions. Generally, microscopic
examination of acridin orange/ethidium bromide stained cells can be recommended as the most reliable of the methods tested.
2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
Keywords: Apoptosis; Method; Microscopy; Cycloheximide; Leukocytes

1. Introduction
Apoptosis is a well-controlled, tightly-regulated physiological process, in which the cells participate in self-destruction.
A large body of evidence suggests that apoptosis is a central
mechanism in embryogenesis and morphogenesis, immune

Abbreviations: CHX, cycloheximide; PBMC, peripheral blood mononuclear cells; PMNC, polymorphonuclear cells; CLL, chronic lymphocytic leukemia; AO/EB, acridine orange/ethidium bromide; AnnV/PI, annexin V/
propidium iodide.
* Corresponding author. Tel.: 381 34 331344; fax: 381 34 331345.
E-mail address: dejan.baskic@gmail.com (D. Baskic).

system regulation, hematopoesis and control of normal tissue

turnover (Vaux and Korsmeyer, 1999), but it has been also
implicated in a variety of diseases (Arends and Wyllie,
1991). Failure of cells to undergo normal apoptotic cell death,
or increased cell loss by apoptosis, may be involved in the
pathogenesis of cancer, autoimmune disorders, neurodegenerative disorders, AIDS and myelodisplastic syndromes.
Apoptosis, or programmed cell death, is essential for
the normal development, homeostasis and function of the immune system (JJ Cohen et al., 1992; Golstein et al., 1991).
Leukocyte apoptosis must be tightly controlled to allow
normal lymphocyte differentiation and neutrophil function in
inflammation, and to prevent malignancy and autoimmunity

1065-6995/$ - see front matter 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.cellbi.2006.06.016

D. Baskic et al. / Cell Biology International 30 (2006) 924e932

(Krammer et al., 1994). Leukocytes removed from their natural environment are reported to undergo apoptosis. However,
little apoptosis occurs in peripheral blood mononuclear cells
(PBMC) after 24 h culture in medium (Gupta et al., 1998),
whereas peripheral blood-derived polymorphonuclear cells
(PMNC) undergo rapid apoptosis during this period (Brach
et al., 1992). Although apoptosis can occur spontaneously,
the apoptotic pathway in leukocytes can also be activated by
signals arising from various cell-physiological conditions, by
external stimuli that trigger surface receptors (Schulze-Osthoff
et al., 1998; Suda et al., 1997), or by drugs such as glucocorticoids (Brunetti et al., 1995), antitumor agents such as actinomycin D, doxorubicin and camptothecin (Nagami et al., 2002)
and cytotoxic compounds including cycloheximide (Lemaire
et al., 1999).
Macromolecule synthesis was originally considered essential for active cell death, but it now appears that synthesis inhibitors can either stimulate or prevent apoptosis, depending
on the cell system used (Marcu et al., 1999; Columbano
et al., 1992). For instance, cycloheximide is known to induce/stimulate or to inhibit/prevent apoptosis depending on
the cell type (Tessitore et al., 1999; Collins et al., 1991;
Lemaire et al., 1999).
Since apoptosis is involved in an ever-growing number of
physiological and pathological processes, reliable methods
for detecting cell death (apoptosis and necrosis) are essential.
Several sensitive methods for detecting apoptosis have been
developed, based on the different morphological or biochemical features of apoptosis and necrosis.
In this study, we compared several methods for detecting
apoptosis and necrosis in human leukocytes treated with cycloheximide, as a model of induction of cell death.
2. Materials and methods
2.1. Cell preparation and culturing
Heparinized blood (10 ml) was obtained by venipuncture from 10 healthy
volunteers. Peripheral blood mononuclear and polymorphonuclear cells were
separated by single step continuous density-gradient centrifugation with Histopaque 1077 (Sigma, Germany). The separated cells were washed three times
with culture medium RPMI 1640 (Sigma, Germany) and finally suspended in
RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin G and 100 mg/ml streptomycin. Cell number and viability were determined using trypan blue and acridine orange/ethidium bromide staining (all
from Sigma, Germany). In some experiments, PBMC from patients with
chronic lymphocytic leukemia (CLL) were used.

2.2. Induction of cell death

Apoptosis was induced by incubating the cells (3  106/ml) with various
doses (2 mg/ml, 20 mg/ml, 200 mg/ml and 2000 mg/ml) of cycloheximide (Oncogene, Germany) for up to 24 h at 37  C in 5% CO2. In some experiments,
cell death was induced by UVB. Briefly, the cell suspension (3  106 cells/
ml) was transferred to 33 mm Petri dishes (Spectar, SCG), placed over
a UV transilluminator (MacroVue UV-20, Hoefer) and exposed to 312 nm radiation (UVB) at room temperature for 5 s, 15 s, 30 s, 60 s, 5 min, 15 min,
30 min or 60 min. The cultures were subsequently incubated for 2 h or 24 h,
respectively, under standard conditions (37  C in 5% CO2). After cultivation,
the cells were harvested by centrifugation, suspended in PBS (pH 7.2) and
aliquoted for staining procedures and electrophoresis.

925

2.3. Light microscopic analysis of cell death

Cells were analyzed by two methods.
1. May GrunwaldeGiemsa staining (Merck, Germany) of methanol-fixed
cytospin preparations. Cells were examined to identify the type of cell
death on the basis of the following morphological criteria:
 Apoptotic cells: nuclear shrinkage and chromatin condensation, cytoplasmic membrane blebbing, reduction of cell volume and formation of apoptotic bodies.
 Necrotic cells: nuclear and cytoplasmic swelling, chromatin flocculation, cytoplasmic and nuclear membrane dissolution or lysis.
2. Trypan blue staining and haemocytometry. To estimate the percentage of
dead cells, staining with 0.4% trypan blue in PBS was used. After 10 min
incubation, the cells (minimum 300) were scored for dye uptake (blue
cells are dead).

2.4. Fluorescence microscopic analysis of cell death

We used two different fluorescent assays.
1. Acridine orange/ethidium bromide (AO/EB) double staining. Acridine orange is taken up by both viable and nonviable cells and emits green fluorescence if intercalated into double stranded nucleic acid (DNA) or red
fluorescence if bound to single stranded nucleic acid (RNA). Ethidium
bromide is taken up only by nonviable cells and emits red fluorescence
by intercalation into DNA. We distinguished four types of cells according
to the fluorescence emission and the morphological aspect of chromatin
condensation in the stained nuclei. (1) Viable cells have uniform bright
green nuclei with organized structure (PMNCs also have orange cytoplasm). (2) Early apoptotic cells (which still have intact membranes but
have started to undergo DNA cleavage) have green nuclei, but perinuclear
chromatin condensation is visible as bright green patches or fragments.
(3) Late apoptotic cells have orange to red nuclei with condensed or fragmented chromatin. (4) Necrotic cells have a uniformly orange to red nuclei with organized structure. One microlitre of dye mixture (100 mg/ml
AO and 100 mg/ml EB in distilled water) was mixed with 9 ml of cell suspension (0.5  106 cells/ml) on a clean microscope slide. The suspension
was immediately (fast uptake) examined by fluorescence microscopy
(Polywar, Reinchard Jung) at 400 magnification. A minimum of 300
cells was counted in every sample.
2. Annexin V/propidium iodide (AnnV/PI) staining. This assay is based on
the ability of the protein annexin V to bind to phosphatidylserine (PS) exposed on the outer membrane leaflet in apoptotic cells (PS also appears
on the necrotic cell surface). In viable cells, PS is located in the inner
membrane leaflet, but upon induction of apoptosis it is translocated to
the outer membrane leaflet and becomes available for annexin V binding.
The addition of PI enabled viable (AnnVneg/PIneg), early apoptotic
(AnnVpoz/PIneg), late apoptotic (AnnVpoz/PIpoz) and necrotic (AnnVneg/
PIpoz) cells to be distinguished. The annexin V assay was performed following the manufacturers instructions (Annexin V-FITC kit, Bender
MedSystems, Austria). Briefly, the cells were washed with PBS and resuspended in prediluted binding buffer (4). The cell density was adjusted to 2e5  106 and 195 ml of cell suspension were mixed with
5 ml Annexin V-FITC and incubated for 10 min in the dark at room temperature. The cells were washed with binding buffer, resuspended in
195 ml binding buffer, counterstained with 10 ml of the 20 mg/ml propidium iodide stock solution (final concentration 1 mg/ml) and examined
under the fluorescence microscope.

2.5. DNA fragmentation: analysis by agarose gel

electrophoresis
This procedure is based on internucleosomal DNA cleavage, a characteristic biochemical hallmark of the apoptotic mode of cell death. When DNA

extracted from apoptotic cells is subjected to gel electrophoresis, a typical

internucleosomal ladder of DNA fragments is produced. Briefly, cellular
DNA was extracted from human PBMC and PMNC. The cells were lysed
with 1% SDS in TE buffer (10 mM Tris, pH 8.0, 0.5 mM EDTA, 1% sodium dodecyl sulfate) and digested with proteinase K (100 mg/ml; Oncogene, Germany) for 4 h at 56  C. The samples were extracted with phenol
and chloroform and the DNA was precipitated with a 1/10 volume of 3 M
sodium acetate and an equal volume of ethanol, pelleted at 13,000  g,
and resuspended in TE buffer and 10 mg/ml of DNase-free RNase (Oncogene, Germany) for 30 min at 37  C. The DNA samples were analyzed on
2% agarose gels (Agarose, Eurogentec, Belgium) in TAE buffer (0.04 M
Tris-acetate, 2 mM EDTA) by electrophoresis at 50 V for 120 min. The
DNA was stained with EB (0.5 mg/ml) and visualized using 312 nm UV
radiation.

2.6. Statistics
Statistical analyses were performed using commercially available software
(SPSS version 8.0; SPSS Inc., Chicago, IL, USA). The distributions of data
were evaluated for normality using the KolmogoroveSmirnov test and then
retested using a chi-square test. For nonparametric variables, differences
between two independent groups were determined by the ManneWhitney
U-test. Comparisons between three or more groups of nonparametric variables
were performed by the KruskaleWallis test. A P-value < 0.05 from two-sided
tests was considered statistically significant.

3. Results
3.1. Different effects of CHX on the induction of
apoptosis in PBMC and PMNC
Freshly-prepared PBMC showed minimal apoptosis after
24 h culture. In contrast, peripheral blood-derived PMNC do
not survive more than 24e48 h in vitro without the addition
of survival-promoting cytokines. In order to determine
whether inhibition of protein synthesis affected spontaneous
apoptosis, human PBMC and PMNC were cultured in the absence or presence of 200 mg/ml CHX, high above the concentration shown to completely inhibit protein synthesis
(Frankfurt et al., 1994). Fig. 1 shows that CHX increased
the percentage of apoptotic PBMC (AO/EB: 14% vs 7% in
control; AnnV/PI: 16.9% vs 11.7% in control). However,
CHX did not increase spontaneous apoptosis in PMNC. Indeed, CHX had no effect on PMNC apoptosis; it failed to protect these cells against spontaneous apoptosis, which remained
at high levels (AO/EB: 88.3% vs 89.4% in control; AnnV/PI:
89.3% vs 86.9% in control).
3.2. Dose-response study of effect of CHX on PBMC
apoptosis
Since PBMC appeared to be sensitive to CHX-induced
apoptosis, a dose-response study was performed. Compared
to control values (AnnV/PI: 11.7%; AO/EB: 7%), the percentage of apoptotic PBMC was slightly increased at lower
concentrations of CHX (AnnV/PI: 11.6%; AO/EB: 9.3% at
2 mg/ml and AnnV/PI: 13.9%; AO/EB: 11.1% at 20 mg/ml)
and markedly enhanced at higher concentrations (AnnV/PI:
16.9%; AO/EB: 14% at 200 mg/ml), with the maximal

Percentage of apoptotic cells

D. Baskic et al. / Cell Biology International 30 (2006) 924e932

100%
80%
60%
40%
20%
0%

PBMC

PMNC

PBMC

AO/EB

PMNC

AnnV/PI

Control

0.07

0.89

0.12

0.87

CHX 200g/ml

0.14

0.88

0.17

0.89

Fig. 1. Comparison of the number of apoptotic cells scored by fluorescence microscopy using acridine orange/ethidium bromide (AO/EB) and annexin V/
propidium iodide (AnnV/PI). Normal human PBMC and PMNC were cultured
in the presence or absence of 200 mg/ml cycloheximide (CHX). An increase in
the number of apoptotic cells was observed in cycloheximide-treated PBMC.
The results represent the means of three independent experiments.

response at 2000 mg/ml resulting in 52.5% (AnnV/PI) and

49.3% (AO/EB) of apoptotic PBMC (Fig. 2).
3.3. Methodological aspects of apoptosis detection
3.3.1. Light microscopy
3.3.1.1. May GrunwaldeGiemsa staining. Cycloheximidetreated PBMN and PMNC showed morphological features
of apoptosis. Striking apoptotic cellular changes observed
by light microscopy included cell shrinkage, masses of
condensed chromatin adjacent to the nuclear envelope,
membrane blebbing and cytoplasmic and nuclear fragmentation leading to the formation of apoptotic bodies. However,
a small but constant percentage of necrotic cells was

Percentage of apoptotic cells

926

60%
50%
40%
30%
20%
10%
0%

control

20

200

2000

CHX g/ml
AnnV/PI

0.12

0.12

0.14

0.17

0.53

AO/EB

0.07

0.09

0.11

0.14

0.49

Fig. 2. Dose-response curves for cycloheximide (CHX)-induced apoptosis in

PBMC measured by fluorescence microscopy using acridine orange/ethidium
bromide (AO/EB) and annexin V/propidium iodide (AnnV/PI). Normal human
PBMC were cultured in the presence or absence of different concentrations of
CHX. A dose-dependent increase in the number of apoptotic cells was
observed in cycloheximide-treated PBMC, with a maximal response at
2000 mg/ml resulting in 52.5% (AnnV/PI) and 49.3% (AO/EB) of apoptotic
PBMC. The curves are the averages of seven independent experiments.

D. Baskic et al. / Cell Biology International 30 (2006) 924e932

3.3.2. Fluorescence microscopy

Cycloheximide-treated PBMC and PMNC were stained
with AO/EB and AnnV/PI and analyzed under a fluorescence
microscope for the percentage of viable, early and late apoptotic and necrotic cells.
3.3.2.1. Acridine orange/ethidium bromide (AO/EB) double
staining. The results obtained with AO/EB double staining
are represented in Fig. 4. Compared with the spontaneous apoptosis observed in control cells (early apoptotic 3%; late apoptotic 4%), PBMC treated with low doses of CHX showed
slightly increased percentages of early apoptotic (2 mg/ml:
3.7%; and 20 mg/ml: 3.6%;), and late apoptotic (2 mg/ml:
100%

Viability

80%
60%
40%
20%
0%

0'

5"

15"

30"

60"

5'

15'

30'

60'

UVB 312nm
0.94

0.94

0.93

0.86

0.81

0.79

0.25

0.25

0.25

AO/EB 0.93

0.93

0.94

0.87

0.68

0.6

0.2

0.15

TB

Fig. 3. Comparison of percentage of viable PBMC irradiated with UVB measured by trypan blue (TB) and acridine orange/ethidium bromide (AO/EB)
staining. A cell suspension (3  106 cells/ml) was transferred to 33 mm Petri
dishes, placed over an UV transiluminator and exposed to 312 nm radiation
(UVB) at room temperature, for various times (5 s, 15 s, 30 s, 60 s, 5 min,
15 min, 30 min and 60 min). The cultures were subsequently incubated for
2 h under standard conditions (37  C in 5% CO2). Loss of cell viability is
dose and time dependent, but the percentage of live cells measured with TB
at given irradiation times is higher than that measured with AO/EB. The results
represent the means of three independent experiments.

PBMC
60%

40%

20%

0%

control

20

200

2000

CHX g/ml
EA

0.03

0.04

0.04

0.06

0.34

LA

0.04

0.06

0.08

0.08

0.15

TA

0.07

0.1

0.11

0.14

0.49

b
Percentage of apoptotic cells

3.3.1.2. Trypan blue staining. To compare lymphocyte viability using a membrane-excluded dye (trypan blue) visible by
light microscopy and a combination of membrane-excluded
and nonexcluded dyes (AO/EB) visible by fluorescence microscopy, we measured lymphocyte death following CHX
treatment or UVB irradiation. With increased doses of CHX
(data not shown) or increased duration of irradiation, the number of lymphocytes exhibiting AO staining decreased. The loss
of cell viability was dose and time dependent. The loss of viability as revealed by trypan blue was also dose and time dependent, but the percentage of live cells at given doses or
irradiation times was higher than that measured with AO/
EB. Cells irradiated for 60 min had 0% AO/EB viability (viability zero), values never observed with trypan blue staining.
The results are shown in Fig. 3.

a
Percent of apoptotic cells

observed in all samples examined. Necrotic cells show cytoplasmic vacuolization, nuclear swelling and rupture of both
nuclear and plasma membranes, resulting in decreased definition of cellular outlines.

927

PMNC

100%
80%
60%
40%
20%
0%

EA

LA

TA

control

0.8

0.08

0.88

CHX 200 g/ml

0.83

0.06

0.89

Fig. 4. Different subsets of apoptotic cells as percentages of all cells measured

by AO/EB fluorescence staining. (a) PBMC treated with CHX showed a dosedependent increase in percentage of early (EA) late (LA) and total (TA)
apoptotic cells. (b) Compared to control, CHX-treated PMNC showed similar
percentages of early, late and total apoptotic cells. The curves are the averages
of seven independent experiments.

6.1%; and 20 mg/ml: 7.6%;) cells, with concomitant increase

in the total number of apoptotic cells (2 mg/ml: 9.8% vs 7%;
and 20 mg/ml: 11.2% vs 7%). Higher CHX doses, 200 mg/ml
and 2000 mg/ml, significantly increased apoptosis in PBMC,
with early apoptotic cells predominating (6.4% and 34.3%)
and late apoptotic cells representing 7.6% and 15%; 14%
and 49.3% of all cells were apoptotic, respectively. As mentioned above, CHX has no effect on PMNC apoptosis, since
both control and CHX-treated PMNC showed similar percentages of early (80.7 vs 82.7), late (8.7 vs 5.6) and total (89.4 vs
88.3) apoptotic cells. The percentage of necrotic cells remained approximately constant at a low level, independently
of the CHX concentration. Fig. 6 (see later) shows the different morphologies of cells stained with AO/EB, classified as
described in Section 2.
3.3.2.2. Annexin V/propidium iodide (AnnV/PI) staining.
AnnV/PI staining was also used to identify apoptotic cells
(Fig. 5). The results obtained by this method were similar to
those obtained with the AO/EB staining procedure. However,
the numbers of early apoptotic cells differed somewhat.
Compared to the control value (5%), the percentage of early

D. Baskic et al. / Cell Biology International 30 (2006) 924e932

Percentage of apoptotic cells

928

PBMC
60%

40%

20%

0%

control

20

200

2000
0.31

CHX g/ml
EA

0.05

0.03

0.05

0.08

LA

0.07

0.09

0.1

0.09

0.22

TA

0.12

0.12

0.14

0.17

0.53

Percentage of apoptotic cells

PMNC

100%
80%
60%
40%
20%
0%

EA

LA

TA

control

0.79

0.08

0.87

CHX 200 g/ml

0.81

0.08

0.89

Fig. 5. Different subsets of apoptotic cells as percentages of all cells measured

by AnnV/PI fluorescence staining. (a) Cycloheximide at the given concentrations (except 2 mg/ml) dose-dependently increases the percentage of earlyAnnVpos/PIneg,(EA) late-AnnVpos/PIpos (LA) and total (TA) apoptotic
PBMC. (b) Compared to control, PMNC treated with 200 mg/ml CHX showed
equal numbers of total, early and late apoptotic cells. The curves are the averages of seven independent experiments.
pos

neg

apoptotic cells (AnnV /PI ) was reduced by 2 mg/ml CHX

(2.7%), but significantly increased by 200 mg/ml (8%) and
2000 mg/ml (31.1%). However, as observed with AO/EB staining, cycloheximide increased the number of late (AnnVpos/
PIpos) and total apoptotic cells and this increase was dosedependent over the concentration range 2e2000 mg/ml, resulting in 8.9%e22.1% late apoptotic and 11.6%e53.2% total
apoptotic cells, respectively. On the other hand, there was no
difference in apoptosis between CHX-treated PMNC and control samples. As demonstrated in Fig. 5, the control PMNC
cultures contained 86.9% apoptotic cells after 24 h, with early
apoptotic cells predominating (78.9%) and late apoptotic cells
representing 8% of the total. Peripheral blood PMNC treated
with 200 mg/ml CHX showed equal numbers of total
(89.3%), early (81.4%) and late (7.9%) apoptotic cells.
3.4. Agarose gel electrophoresis
Chromatin degradation into 180e200 bp fragments is
commonly associated with apoptosis in many cell types. In
order to investigate whether the morphological changes

Fig. 6. Various pictures of apoptotic cells stained with AO/EB. The morphological patterns of apoptotic cells are described in the text.

observed in CHX-treated or UVB-irradiated PBMC and

PMNC were consistent with chromatin degradation, DNA extracted from apoptotic cells was electrophoresed on 2% agarose gels. As shown in Fig. 7, fragmented DNA was barely
detectable or nonexistent at any CHX dose or irradiation
time. However, substantial amounts of high-molecular-weight
DNA were present in some samples, indicating that either
a small subset of cells had undergone internucleosomal
DNA digestion or that only a fraction of each cells DNA
had become fragmented. Although DNA fragmentation has
been seen in many cell types, and is generally considered
the biochemical hallmark of apoptosis, it may be delayed,
partial or absent in some cell types or experimental conditions. As observed by others, chronic lymphocytic leukemia
cells are characterized by an extended lifespan in vivo
(Kipps, 2000), but are known to be significantly more sensitive to spontaneous (in vitro-culture-induced) or drug-induced
apoptosis (Panayiotidis et al., 1993; Vilpo et al., 2000).
Therefore, CHX-treated PBMC from chronic lymphocytic
leukemia (CLL) patients were assayed for fragmented
DNA. As shown in Fig. 7, PBMC from CLL patients treated
with 200 mg/ml and 2000 mg/ml CHX showed similar
DNA laddering patterns following electrophoresis. This result

D. Baskic et al. / Cell Biology International 30 (2006) 924e932

929

late stage of apoptosis and various types of late apoptotic

(dead) cells with condensed shrinked nuclei are shown on
Fig. 6c and d. Fig. 6e shows viable PMNC. Fig. 6f shows
viable apoptotic PMNC with condensed and fragmented nuclei
and Fig. 6g shows late apoptotic PMNC with condensed
nuclei.
AnnV/PI staining readily identified apoptotic cells by an
integral membrane staining of FITC-labeled AnnV. In the
absence of membrane integrity condensed chromatin stained
red by intercalating day PI (Fig. 8).
Since it could not be assumed that the features compared
were normally distributed, the nonparametric KruskaleWallis
test was chosen to evaluate the statistical significance of the
data in Table 1. To determine whether the differences in apoptotic cell numbers were statistically significant in each
staining method, the number of cells positively stained
with AnnV/PI and the number of apoptotic cells stained
with AO/EB obtained from seven culture series were compared by the KruskaleWallis and Spearman rank correlation
tests. As shown in Table 1, the results obtained were highly
reproducible, and there were no differences between the two
methods.

4. Discussion

Fig. 7. DNA electrophoresis of CHX-treated PBMC, PMNC and CLL cells.

Normal human PBMC and PMNC, and CLL cells, were cultured in the presence or absence of 200 mg/ml and 2000 mg/ml cycloheximide (CHX). Lane A,
molecular weight marker. Lane B, PBMC exposed to 200 mg/ml CHX. Lane C,
PBMC exposed to 2000 mg/ml CHX. Lane D PMNC exposed to 200 mg/ml
CHX. Lane E, CLL cells exposed to 200 mg/ml CHX. Lane F, CLL cells
exposed to 2000 mg/ml CHX.

demonstrated that PBMC and PMNC apoptosis, as well as

apoptosis following CHX or UV treatment of CLL PBMC,
revealed by morphology, AO/EB and AnnV/PI staining do
not correlate with DNA fragmentation.
3.5. Comparison of the fluorescence methods applied to
detect apoptosis
When different approaches are used to detect apoptosis in
a cell population, it is reasonable to compare their efficacies.
To illustrate the results obtained using AO/EB staining, photomicroscopic images of the cultures are presented. Fig. 6a
shows normal viable PBMC (green). Various pictures of apoptotic PBMC selected from other cultures are given in
Fig. 6bed. An apoptotic cells in its earliest stage possessed
a condensed or shrunk nucleus with perinuclear chromatin
condensation which appeared as a bright green arcs
(Fig. 6b); Viable apoptotic cells with fragmented nuclei, apoptotic cell with condensed, yellow/orange nuclei, entering the

The objective of this study was to compare several different

methods for measuring spontaneous (culture-induced) and
CHX-induced apoptosis in peripheral blood leukocytes.
A number of reports indicate that induction of apoptosis
requires de novo protein synthesis (Ucker et al., 1989) and
therefore CHX protein synthesis inhibitor should, antagonize
spontaneous and drug-induced apoptosis in different cell types
(Marcu et al., 1999; Tessitore et al., 1999). The protective effect of CHX was initially attributed to inhibition of death gene
expression. However, another hypothesis is that the death
machinery is pre-existent and must be restrained by the continuous synthesis of short-lived protective molecules. This hypothesis predicts that CHX, as a protein inhibitor, should
induce rather than inhibit apoptosis. Indeed, some earlier reports indicated that CHX treatment augmented apoptosis in
different cell types (Columbano et al., 1992; Collins et al.,
1991). Similarly, Lemaire and associates observed that low
doses (2.5 mg/ml) of CHX stimulated B cell apoptosis (Lemaire et al., 1999). Interestingly, the same authors found that
lowering the concentration of CHX led to the opposite effect:

Fig. 8. AnnV/PI staining of the apoptotic cell. An image of PBMC showing:

(a) early (AnnVpos/PIneg), (b) late (AnnVpos/PIpos) apoptotic and (c) necrotic
(AnnVneg/PIneg) cell.

D. Baskic et al. / Cell Biology International 30 (2006) 924e932

930

Table 1
Percentages of early (EA), late (LA) and total (TA) apoptotic cells measured with AnnV/PI and AO/EB fluorescence microscopy

EA

Control

2 mg CHX

20 mg CHX

200 mg CHX

2000 mg CHX

KruskaleWallis

Spearman

AoEb

3.7

3.6

6.4

34.3

Anex

2.7

4.7

31.1

H 2.58,
p > 0.05
H 1.96,
p > 0.05

r 0.243,
p > 0.05
r 0.227,
p > 0.05

AoEb

c2 2.987,
p > 0.05
4

c2 2.054,
p > 0.05
6.1

c2 3.01,
p > 0.05
7.6

c2 3.245,
p > 0.05
7.6

c2 2.67,
p > 0.05
15

Anex

6.7

8.9

9.6

8.9

22.1

H 1.69,
p > 0.05
H 2.43,
p > 0.05

r 0.231,
p > 0.05
r 0.248,
p > 0.05

AoEb

c2 2.324,
p > 0.05
7

c2 3.14,
p > 0.05
9.8

c2 2.45,
p > 0.05
11.2

c2 0.384,
p > 0.05
14

c2 2.23,
p > 0.05
49.3

Anex

11.7

11.6

14.3

16.9

53.2

H 1.954,
p > 0.05
H 1.872,
p > 0.05

r 0.193,
p > 0.05
r 0.197,
p > 0.05

c2 2.32,
p > 0.05

c2 1.54,
p > 0.05

c2 1.83,
p > 0.05

c2 2.34,
p > 0.05

c2 2.45,
p > 0.05

Chi-square
LA

Chi-square
TA

Chi-square
Statistical analysis.

CHX at 0.05 mg/ml reduced spontaneous as well as druginduced apoptosis.

Our study has shown that CHX, depending on the cell type
used, augments spontaneous apoptosis in cultured PBMC but
has no effect on PMNC apoptosis, reflecting the opposing
effects of this protein synthesis inhibitor on the induction of
apoptosis. Thus, the dual activity of CHX indicates that the
signaling pathways leading to apoptosis differ from one cell
type to another. Although data on CHX concentration and its
ability to completely inhibit protein synthesis are contradictory, it is noteworthy that high concentrations of CHX
(>200 mg/ml) could induce apoptosis by non-specific metabolic stress. Our results related to CHX-induced apoptosis in
PBMC agree with those obtained by other authors who used
the same model of cell death (Potter et al., 1999; Stein
et al., 2000). However, contrary to the data presented in our
study, most researchers have found that CHX strongly enhances spontaneous apoptosis in PMNC (Chacon-Cruz et al.,
1998; Squier et al., 1999). For example, Sullivan et al.
(1996) found that after 24 h in culture, CHX increased the
number of apoptotic PMNC from 73% to 90% of the initial
cells. Since we were unable to attribute these discrepancies
in results to the mechanism or combination of mechanisms,
accounting for these observed differences remains to be
elucidated.
The present study also evaluated and compared several of the
available methods for detecting apoptosis. These methods exploit the most important morphological and biochemical
changes occurring in apoptotic cells. Unfortunately, no single
assay can be used alone with perfect specificity and sensitivity.
The morphological criteria for apoptosis, condensation of chromatin, cell shrinkage, as well as blebbing, although the oldest,
remains probably the most specific of all assays. Therefore,
most researchers agree that electron microscopy (TEM as
well as SEM) is the only definitive way to identify apoptotic
cells. Since these methods require expensive equipment and

are not practicable in all laboratories, other assays should be

performed first. Analysis of a Giemsa-stained cytospin allows
recognition of the main morphological characteristics of necrotic and apoptotic death. In vitro, however, late stage apoptotic cells undergo secondary necrosis with degenerative changes,
biasing the results towards exaggeration of necrotic cell death.
Moreover, light microscopy identifies features that occur after
major biochemical events, so the time at which apoptosis is analyzed must be optimized. Though widely used for viability
testing, trypan blue staining is valueless for detecting apoptosis.
Staining of apoptotic cells with fluorescent dyes such as AO
and EB is considered the correct method for evaluating the
changed nuclear morphology (Leite et al., 1999; Gasiorowski
et al., 2001; Savitskiy et al., 2003). As demonstrated in Fig. 6,
AO/EB staining, based on nuclear morphology (perinuclear
chromatin condensation, nuclear collapse and eventual fragmentation), allows us to distinguish several subpopulations
of apoptotic cells from viable and necrotic cells. The addition
of the light microscopy appearance of these AO/EB cells, for
example cell rounding, surface blebbing and shrinkage, provide a complete morphological profile of an apoptotic cell.
AnnV staining for both microscopy and flow cytometry is
a widely-used method for detecting apoptosis (Schutte et al.,
1998; Plasier et al., 1999; Vermes et al., 1995). Although
flow cytometry as an operator-independent method has the advantage over manual microscopy, it is limited when cells are
simultaneously stained with AnnV and PI (primary necrosis
or in vitro secondary necrosis of late apoptotic cells). Annexin
V, which binds to PS exposed on the outer membrane leaflets
of apoptotic cells, also appears on the surfaces of dead cells
(Stewart, 1994; Bossy-Wetzel and Green, 2000). In addition,
the characteristics of late apoptosis include some loss of membrane integrity and PI uptake. Therefore, flow cytometry cannot distinguish between late apoptotic and necrotic cells,
which can both be AnnVpos/PIpos. Morphological analysis by
light microscopy and/or AO/EB fluorescence microscopy

D. Baskic et al. / Cell Biology International 30 (2006) 924e932

should resolve this problem. However, on the basis of PI

stained nuclear morphology, fluorescence microscopy of
AnnVpos/PIpos cells allowed us to assign every cell to a defined
category (late apoptotic or necrotic). Both fluorescence
methods (AO/EB and AnnV/PI) provided reliable, reproducible results and distinguished clearly between subpopulations
of apoptotic cells. The apoptotic cell subpopulations identified
by the AO/EB and AnnV/PI methods correlated closely.
Although there is no perfect correspondence/correlation
between the methods tested, we interpreted these findings as
support for the argument that the AO/EB and AnnV/PI
methods give the same results.
Agarose gel electrophoresis was also used to identify apoptotic cells. For cells that fragment their DNA into oligonucleosome-sized multiples, DNA fragmentation analysis by
agarose gel electrophoresis is considered highly specific but
not very sensitive, requiring DNA from a large number of
cells. Some studies have shown that DNA degradation is a relatively late event in the apoptotic cascade and chromatin degradation to large-scale fragments precedes internucleosomal
cleavage (Collins et al., 1992). Moreover, apoptosis-associated
DNA degradation sometimes stops after large DNA fragments
of 30e50 kb are generated and does not proceed to internucleosomal cleavage (GM Cohen et al., 1992). As we have
shown, DNA fragmentation did not correlate in all circumstances with apoptosis measured by all the independent
methods used in this study, namely, light microscopy (Giemsa
staining) and fluorescence microscopy (AO/EB and AnnV/PI
staining). Therefore, although applicable to a wide spectrum
of cell types, agar electrophoresis of extracted DNA cannot
be applied to all cell types and apoptotic conditions.
Finally, in our experience, among the methods used to detect apoptosis, microscopic examination of AO/EB stained
cells can be recommended as the most reliable, since it makes
it possible to perform high-quality studies of cell morphology,
nuclear and chromatin disintegration as well as to distinguish
viable, early or late apoptotic and necrotic cells.

Acknowledgements
This work was supported by grant 1637 from the Ministry
of Science, Technologies and Development, Republic of
Serbia. We thank Dr Nela onovic for statistical expertise.
We also thank Milan Milojevic for administrative, technical
and logistic assistance.

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