COMMUNICATION TO THE EDITOR
Analysis of Apoptosis in FUCCI HeLa Cells
INTRODUCTION
Later stages of apoptotic cell death are characterized
by the fragmentation of cellular DNA (1). For example,
HeLa cells exposed to a lethal dose of UVC irradiation (30
J/m2) undergo apoptosis and exhibit DNA fragmentation
(2). DNA fragmentation, which results in reduced DNA
content, can be assessed using several methods, the most
common being cell analysis by flow cytometry. The stand-
ard measure for the level of apoptosis in response to a
tested treatment is the fraction of cells with sub-G1 DNA
content, indicating that the DNA of these cells underwent
fragmentation and DNA loss (3). However, sub-G1 DNA
content is reached only after a significant amount of cellu-
lar DNA is lost. We predicted that in cells that start under-
going apoptosis at late stages of the cell cycle, in which
DNA content is higher, it would take longer for cellular
DNA content to decrease to the sub-G1 DNA level. In
addition, cells in which apoptosis was triggered in G2/M
would pass through stages of having G2/M, S, and then G1
levels of DNA content. Thus, the population of cells identi-
fied by flow cytometry as containing a G1 level of DNA
may include apoptotic cells in which DNA fragmentation is
incomplete. Ignoring this population of cells may lead to
significant underestimation of the fraction of cells under-
going apoptosis at certain time points. In addition, the
assumption that all cells in the G1 fraction are living may
lead to misinterpretation of the relationship between apo-
ptosis and cell cycling. Indeed, this phenomenon has been
demonstrated in HL60 cells (4,5).
Here, we used HeLa cells stably expressing fluorescent
ubiquitination-based cell cycle indicator (FUCCI) fusion pro-
teins as cell cycle sensors to perform a systematic quantitative
analysis of the kinetics of DNA depletion in cells at different
stages of cell cycle progression and to specifically follow cells
that started undergoing apoptosis in S-G2/M phases (6). We
demonstrate that the longer time required for DNA fragmen-
tation in these cells may lead to misinterpretation of flow cyto-
metry analysis results.
Received 8 February 2011; Revision Received 20 February 2011;
Accepted 22 February 2011
*Correspondence to: Sharon Eden, Department of Developmental
Biology and Cancer Research, IMRIC, The Hebrew University-
Hadassah Medical School, Jerusalem 91120, Israel.
E-mail: sharoned@ekmd.huji.ac.il
Cytometry Part A • 79A: 243 246, 2011
MATERIALS AND METHODS
Cell Culture and Irradiation
HeLa.S-FUCCI cells (6) were grown in DMEM with 10%
fetal bovine serum and penicillin/streptomycin. UVC lamp of
254 nm (Phillips) was used to irradiate cells at 30 J/m2. Immu-
noblots were performed using an anti-GFP (FL) antibody
(Santa-Cruz sc-8334) and antiactin antibody (Hybridoma
bank JLA-20).
Time-Lapse Microscopy
Cells were grown on a Zeiss LMS 710 microscope
equipped with an incubator and X40 Plan-Neofluar lens (NA
0.6). mAG was excited by a 488-nm laser line (Argon laser),
and fluorescence signals were collected at 493–578 nm. mKO2
was excited by a 561-nm laser line (DPSS 561-10) and fluores-
cence signals were collected at 578–696 nm.
Flow Cytometry and TUNEL Assay
Cells were UV irradiated, harvested after the indicated
times, fixed in 75% ethanol, and stored at 220
C. The fixed
cells were rehydrated in PBS on ice for 30 min, and stained
using Apo-BrdU-Red in situ DNA fragmentation assay kit
(BioVision), using Hoechst 33342 instead of 7-AAD. Cells
were analyzed in a BD LSRII (Becton Dickinson) flow cytome-
ter. mAG and BrdU-Red were excited by a 488-nm laser line,
and fluorescence signals were collected at 530 nm (530/30; 505
LP) for mAG and at 575 nm (575/25)(550LP) for BrdU-Red.
Hoechst 33342 was excited by a 355 laser line, and fluores-
cence signals were collected at (450/50). mKO2 fluorescence
was below detection level in these experiments.
RESULTS AND DISCUSSION
To test the possibility that some apoptotic cells may be
mistakenly identified by flow cytometry analysis as live cells,
we exposed HeLa cells to a lethal dose of UV irradiation and
used flow cytometry to analyze their DNA content at different
time points after irradiation. To distinguish between cells in
different phases of the cell cycle, we used HeLa cells stably
Published online 11 March 2011 in Wiley Online Library
(wileyonlinelibrary.com)
DOI: 10.1002/cyto.a.21051
© 2011 International Society for Advancement of Cytometry
COMMUNICATION TO THE EDITOR
expressing FUCCI fusion proteins as cell cycle sensors (6).
These cells express Cdt1-Kusabira Orange fusion protein,
which confers red fluorescence in G1 phase, and Geminin-
Azami Green fusion protein, resulting in green fluorescence in
S, G2, and M phases of the cell cycle. FUCCI cells in cytokin-
esis do not express any fluorescent protein. It has been
reported that Cdt1 is degraded rapidly after UV-induced DNA
damage (7,8). However, the protein domain required for this
degradation was localized to amino acids 1–53 of Cdt1, and
the Cdt1-Kusabira Orange fusion protein contains only amino
acids 30–120 of Cdt1. We verified that the Cdt1-Kusabira
Orange fusion protein is not degraded in HeLa cells after
exposure to UV irradiation by immunoblot with anti-GFP
antibody, which recognizes the Kusabira Orange protein (Fig.
1A). In addition, we followed cell death using time-lapse mi-
croscopy and observed red fluorescence several hours after UV
irradiation in both living and dead cells (Fig. 1B). These data
confirm that the HeLa FUCCI cells should allow us to investi-
gate cell death in S-G2/M phase HeLa cells following UV irra-
diation. Time-lapse microscopy also showed that both red and
green fluorescent cells exhibited apoptotic morphology after
UV exposure and remained fluorescent several hours after they
began to exhibit apoptotic morphology (Fig. 1B).
We used the FUCCI cells to differentiate between cells that
began to undergo apoptosis in S/G2/M and those that began apo-
ptosis in G1. We were particularly interested in the S/G2/M
population, because these cells have higher DNA content and
may take longer to deplete their DNA to sub-G1 DNA levels. We
exposed FUCCI-HeLa cells to UV irradiation and assessed their
DNA content every 2 h using flow cytometry analysis, identifying
the S/G2/M population by gating for green fluorescence. The
nongreen population consists primarily of red-fluorescent cells
in G1 phase, although it also includes a very small fraction of
nonfluorescent cells in cytokinesis. To simplify description of our
results, we will refer to the nongreen cell population as red cells.
As expected, at the time of irradiation, green cells were mainly
gated to the predicted DNA content of S and G2/M phases, and
red cells were primarily gated to G1 DNA content (Fig. 1C). By
16 h postirradiation, 83.9% of the red cells possessed sub-G1
DNA content, but only 11.6% of the green cells had reached sub-
G1 DNA content. These results suggest that cells that started to
undergo apoptosis while in G2/M required a significantly longer
time to deplete their DNA to a sub-G1 level. The percentage of
green cells with G1 DNA content increased from 2 to 16 h after
UV exposure, indicating that cells that became apoptotic in G2/
M went through a period where their DNA content was equiva-
lent to G1-level before it deteriorated to sub-G1 levels. These
results indicate that some of the cells with G1-levels of DNA con-
tent have started undergoing apoptosis.
To confirm that DNA fragmentation is already underway
in the population of green cells with G1-equivalent DNA con-
tent, we performed flow cytometry analysis of cells stained for
fragmented DNA using a TUNEL assay adapted for flow cyto-
metry analysis. The harsh fixing conditions required for
TUNEL staining resulted in fragile cells, and a very significant
reduction in the fraction of cells with sub-G1 DNA content,
regardless of their DNA content at the time of irradiation. We
244
modified the fixation conditions to maintain cell integrity (see
methods). Using this new approach, we observed TUNEL-
positive cells with sub-G1 DNA content 4 h after UV exposure
as expected (Fig. 1C). The peaks of TUNEL-positive cells cor-
responded to populations of cells with different DNA content:
the largest peak of TUNEL-positive cells had sub-G1 level
DNA content, whereas another peak had approximately S-level
of DNA content, similar to the major peak of cells with green
fluorescence at this time point. By 16 h, all peaks of cells with
either green or red fluorescence also stained positive for
TUNEL despite the fact that the DNA content of most of the
green cells remained higher than sub-G1. This observation fur-
ther supports the idea that DNA content alone is not an accu-
rate indicator of cell cycle phase or cell survival in cells
exposed to apoptosis-inducing agents. It also confirms that
not all apoptotic cells appear in the sub-G1 fraction.
This experiment also allowed us to compare the
kinetics of DNA degradation in cells that began apoptosis
during S/G2/M phases to cells that became apoptotic in G1.
Throughout the experiment, the green cells exhibited faster
kinetics of apoptosis, as indicated by TUNEL staining, than
cells that started undergoing apoptosis in G1 (red cells).
Four hours after UV exposure, only 12.7% of the cells with
red fluorescence were TUNEL positive, whereas 27.5% of
the green cells were TUNEL positive. This discrepancy was
observed for the duration of the analysis, further indicating
that cells with higher DNA content start undergoing apo-
ptosis earlier than cells in G1 phase.
Despite the more rapid kinetics, the green cells took lon-
ger to reach sub-G1 DNA content. Sixteen hours after UV irra-
diation, only 55.3% of total cells had sub-G1 DNA content, but
86.1% of all cells were TUNEL positive, demonstrating that
using sub-G1 content as a marker for apoptosis results in a sig-
nificant underestimate. Comparing the proportion of red and
green cells with TUNEL staining revealed that this discrepancy
is due primarily to the longer time course of DNA loss in cells,
which begin apoptosis in S2/G/M. Sixteen hours after irradia-
tion, 76.9% of red cells stained positive for TUNEL, and a sim-
ilar percentage (83.9%) of red cells presented sub-G1 DNA
content. At the same time point, 92.4% of the green cells
stained positive for TUNEL, but only 11.6% exhibited sub-G1
DNA content. In addition, DNA content deficit cannot be used
as a sole marker of apoptotic cells. Mechanical cell degradation
and lysis of mitotic cells or micronuclei may appear as apopto-
tic cells. In addition to DNA deficit, other parameter should be
used to positively identify apoptotic cells (9). We conclude that
measuring the fraction of cells with sub-G1 DNA content is
useful for determining whether certain treatments induce apo-
ptosis but is not an accurate quantitative measure of apoptosis
and can be misleading regarding the relationship between apo-
ptosis and cell cycle progression.
Combining flow cytometry analysis with TUNEL analysis
did not resolve this issue because TUNEL staining labels only
cells in late stages of apoptosis, albeit earlier than DNA dete-
rioration following fragmentation. Our results also indicate
that DNA fragmentation in cells with higher DNA content
occurs with faster kinetics than in cells with G1-level DNA con-
Communication to the Editor
 COMMUNICATION TO THE EDITOR

Figure 1. Analysis of DNA content in UV-exposed FUCCI HeLa cells. (A) Cdt1-Kusabira Orange is stable after UV irradiation. FUCCI HeLa
cells were exposed to 30 J/m2 UVC irradiation, lysed at the indicated time points, and the cell lysates were subjected to immunoblot analy-
sis using anti-GFP antibody and anti-beta-actin antibody as a loading control. Cdt1-Kusabira Orange fusion protein was detected at 34Kd,
and no degradation was observed 2 h after UV irradiation. (B) FUCCI HeLa cells maintain fluorescence after UV irradiation. FUCCI HeLa
cells were exposed to 30 J/m2 UVC irradiation and visualized by time-lapse microscopy. Frames of the indicated time points are shown.
Cells with both red and green fluorescence maintain their color after they exhibit apoptotic morphology. (C) Analysis of DNA content in
UV-exposed FUCCI HeLa cells. FUCCI HeLa cells were exposed to 30 J/m2 UVC irradiation. The cells were labeled for TUNEL staining, and
the DNA was stained with Hoechst 33342. Flow cytometry analysis of HeLa cells after exposure to UV irradiation at the indicated time
points after irradiation is shown. Green, cell with green fluorescence; red, cells with red fluorescence; blue, TUNEL positive cells.

tent. Although the apoptotic process can be followed using
other markers, the different kinetics exhibited by cell popula-
tions in different cell cycle phases will skew the results regard-
less of the method used to identify apoptotic cells. We propose
that the most accurate method of measuring the level of apo-
ptosis is to follow the population of cells in each cell cycle
phase separately as demonstrated here. In addition, because
the kinetics of DNA fragmentation is dependent on cell cycle
phase, the cell density and the fraction of dividing cells should
be taken into account when analyzing cell death by flow cyto-
metry. This is of particular importance when studying apopto-
sis in the context of cell cycle progression. Similar conclusions
were presented in a recent work, demonstrating that combin-
ing cytometry analysis with the FUCCI probe may contribute
to development of an assay that integrates the complexity of
cell cycle regulation into drug discovery screens (10).

Cytometry Part A  79A: 243 246, 2011 245

COMMUNICATION TO THE EDITOR
The authors thank Dr. Atsushi Miyawaki for the RCB2812
HeLa.S-FUCCI cell line, Dan Lehman and Zohar Yagil for
technical assistance, and Eitan Shaulian for careful reading of
the manuscript. The JLA20 hybridoma antibody was obtained
from the Developmental Studies Hybridoma Bank.
Yuval Malka
Sharon Eden*
Humphrey Center and Department of
Developmental Biology and Cancer Research,
IMRIC The Hebrew University-Hadassah Medical
School Jerusalem 91120, Israel
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Communication to the Editor