European Journal of Cell Biology 91 (2012) 413–419

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European Journal of Cell Biology

journal homepage: www.elsevier.de/ejcb

Enrichment of cell populations in metaphase, anaphase, and telophase by

synchronization using nocodazole and blebbistatin: A novel method suitable for
examining dynamic changes in proteins during mitotic progression
Yuki Matsui, Yuji Nakayama ∗ , Mai Okamoto, Yasunori Fukumoto, Naoto Yamaguchi ∗
Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Mitosis is a continuous process to separate replicated chromosomes into two daughter cells through
Received 27 June 2011 prophase, metaphase, anaphase, and telophase. Although a number of methods have been established
Received in revised form to synchronize cells at different phases of the cell cycle, it is difficult to synchronize cells at the specific
22 December 2011
phases, anaphase and telophase, during mitosis because of the short duration of anaphase. Here, we show
Accepted 28 December 2011
that HeLa S3 cells in anaphase and in telophase are successfully enriched by treatment with a combination
of low concentrations of the microtubule-depolymerizing agent nocodazole and the myosin II inhibitor
Keywords:
blebbistatin. After 9-h release from thymidine block at G1/S phase, addition of nocodazole at 20 ng/ml but
Cell cycle synchronization
Blebbistatin
not 40 ng/ml ensures rapid release from the nocodazole arrest. Subsequently, the cells are cultured in the
Anaphase presence of 50 ␮M blebbistatin for 20 and 50 min to enrich cells in anaphase and telophase, respectively.
Nocodazole Western blot analysis verifies down-regulation of phospho-histone H3-Ser10, phospho-Aurora A/B/C, and
Mobility shift cyclin B1 during M-phase progression. Furthermore, we show how the electrophoretic mobility shifts of
c-Yes the Src-family kinases c-Yes and c-Src can change in each phase of mitosis. These results provide a useful
synchronization method for biochemically examining protein dynamics during M-phase progression.
© 2012 Elsevier GmbH. All rights reserved.

Introduction include (i) mitotic shake-off (Tobey et al., 1967), (ii) treatment
with microtubule toxins (Taylor, 1965; Zieve et al., 1980), (iii)
Mitosis is a continuous process to separate the replicated chro- release from the thymidine block, and (iv) release from the G2/M
mosomes into two daughter cells. There are four phases of mitosis: arrest induced by the reversible CDK1 inhibitor RO-3306 (Vassilev
prophase, metaphase, anaphase and telophase. M-phase progres- et al., 2006). However, it is difficult to scrutinize the biochemi-
sion is mainly regulated by post-translational mechanisms, such as cal events occurring during M-phase progression, because of the
phosphorylation and proteolysis. Protein phosphorylation by acti- lack of appropriate methods for cell cycle synchronization through
vation of cyclin B1/CDK1 drives mitotic entry and progression to anaphase and mitotic exit.
metaphase (Nigg, 2001), but cyclin B1 degradation and inactiva- In this study, we explored a method for cell cycle syn-
tion of CDK1 drives mitotic exit and cytokinesis (Clute and Pines, chronization during M-phase progression suitable for examining
1999). In addition, the vast majority of mitotic phosphorylations are biochemical changes in proteins. We show that cell populations
removed upon mitotic exit by mitotic phosphatases (Bollen et al., are efficiently enriched in metaphase, anaphase and telophase by
2009). synchronization using blebbistatin and nocodazole.
Studies examining cell cycle regulatory mechanisms require
cell cycle synchronization of cell populations, and many synchro- Materials and methods
nization methods have been established to synchronize cells at
specific phases of the cell cycle (Davis et al., 2001; Merrill, 1998). Reagents
Cells can be synchronized in M-phase by several methods, which
Nocodazole (Sigma) and blebbistatin (Toronto Research Chem-
icals) were made in stock solution in dimethyl sulfoxide (DMSO).

∗ Corresponding authors at: Department of Molecular Cell Biology, Graduate

Cells
School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba
260-8675, Japan. Tel.: +81 43 226 2869/2868; fax: +81 43 226 2869/2868.
E-mail addresses: nakayama@p.chiba-u.ac.jp (Y. Nakayama), Human epithelial HeLa S3 cells (Japanese Collection of Research
nyama@p.chiba-u.ac.jp (N. Yamaguchi). Bioresources, Osaka, Japan) were cultured in Iscove’s modified

0171-9335/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ejcb.2011.12.008
414 Y. Matsui et al. / European Journal of Cell Biology 91 (2012) 413–419

Dulbecco’s medium (IMDM) supplemented with 5% bovine serum in PBS containing 4% paraformaldehyde for 20 min. The number
(BS). Human breast cancer MCF-7 cells were cultured in IMDM of round-shaped mitotic cells was counted under a phase-contrast
supplemented with 5% fetal bovine serum (FBS). Human colorec- microscope.
tal carcinoma HCT116 cells were cultured in IMDM supplemented
with 1% FBS plus 4% BS.
Cell cycle synchronization

Antibodies
HeLa S3 cells were treated with 4 mM thymidine for 24 h,
washed and released into thymidine-free fresh medium. After
The following antibodies were used: Phospho-Aurora A
9-h release from S-phase arrest, the cells were incubated with
(Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11, Cell Sig-
20–40 ng/ml nocodazole for 4 h. After nocodazole incubation,
naling Technology), IAK1 (4/IAK1, BD Biosciences), AIM-1 (6/AIM-1,
approximately 80% of total cells had mitotic round shapes, and
BD Biosciences), phospho-histone H3 (Ser10) (6G3, Cell Signal-
mitotic cells were collected by mitotic shake-off. The mitotic cells
ing Technology), histone H3 (C-16, Santa Cruz Biotechnology),
were washed to remove nocodazole and then incubated in drug-
cyclin B1 (H-433, Santa Cruz Biotechnology), ␣-tubulin (YOL1/34,
free fresh medium in suspension culture. After 20-min release from
Serotec), hsMad2 (H57520, Transduction Laboratories), Src (327,
nocodazole arrest, cells were incubated with 50 ␮M blebbistatin,
Oncogene Research; GD11, Millipore), Yes (clone1, BD Biosciences),
fixed every 10 min with 4% paraformaldehyde, and stained for ␣-
phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10, Cell Sig-
tubulin and DNA as described above.
naling Technology) and ERK2 (C-14, Santa Cruz Biotechnology).
Horseradish peroxidase (HRP)-F(ab )2 of anti-mouse IgG, anti-
rabbit Ig and anti-rat IgG secondary antibodies (GE Healthcare Results and discussion
Japan) and HRP-F(ab )2 of anti-goat IgG secondary antibody
(Southern Biotechnology Associates, Inc.) were used. Fluorescein To enrich cell populations in a step during M-phase progres-
isothiocyanate (FITC)-conjugated F(ab )2 anti-rat IgG secondary sion, prometaphase arrest and subsequent release are often carried
antibody was obtained from Sigma. out using the microtubule-depolymerizing agent nocodazole. How-
ever, prolonged exposure of cells to nocodazole is known to affect
Immunofluorescence cell-cycle progression from M phase to G1 phase despite extensive
washing, resulting in a reduction of cell synchrony (Zieve et al.,
Immunofluorescence staining was performed as described 1980). To circumvent this adverse drug reaction, a short period of
(Ikeda et al., 2008; Nakayama and Yamaguchi, 2005; Obata et al., nocodazole treatment would be necessary for efficient release from
2010; Yamaguchi and Fukuda, 1995). In brief, cells were fixed in nocodazole arrest and for cell synchronization during M-phase
phosphate-buffered saline (PBS) containing 4% paraformaldehyde progression. However, nocodazole treatment would be continued
for 20 min. Fixed cells were permeabilized and blocked in PBS con- until a sufficient number of cells are arrested at prometaphase
taining 0.1% saponin and 3% bovine serum albumin for 1 h, and then for the following experiment. In this regard, nocodazole should
incubated with a primary and a secondary antibody for 1 h each. be added into cell culture medium just before an increase in the
For DNA staining, cells were subsequently treated with 200 ␮g/ml number of mitotic cells after release from thymidine arrest. To
RNase A for 1 h and 50 ␮g/ml propidium iodide for 30 min. Confo- determine the timing of nocodazole addition and the incubation
cal images were obtained using an LSM510 (Zeiss) laser-scanning period, asynchronous HeLa S3 cells were cultured in the presence
microscope. of 4 mM thymidine for 24 h, and G1/S-phase-arrested cells were
then released in thymidine-free, nocodazole-containing medium.
Western blotting Mitotic cells arrested at prometaphase were quantitated by count-
ing the number of cells showing round-shaped morphology. The
Whole cell lysates were prepared in SDS-sample buffer contain- mitotic index was found to increase during 9–14 h after the release
ing 10 mM Na3 VO4 , 20 mM ␤-glycerophosphate and 50 mM NaF, from thymidine arrest, and most of the cells entered M phase within
subjected to SDS–PAGE and electrotransferred onto polyvinylidene 14 h of culture (Fig. 1A). This result shows that approximately 60%
difluoride membranes (PVDF, Millipore). Immunodetection was of cells may be arrested at prometaphase upon 4-h incubation with
performed as reported previously (Kikuchi et al., 2010; Nakayama nocodazole after 9-h release from thymidine arrest. These results
et al., 2006, 2009; Sato et al., 2009). Sequential reprobing of suggest that nocodazole may be added into cell culture medium at
membranes with a variety of antibodies was performed after 9 h after the release from thymidine arrest and cells may be incu-
the complete removal of primary and secondary antibodies from bated for 4 h to obtain a sufficient number of cells for the following
membranes in stripping buffer or inactivation of HRP by 0.1% experiment.
NaN3 , according to the manufacturer’s instructions. Results were Nocodazole is generally used at 40∼200 ng/ml to accumulate
analyzed using an image analyzer ChemiDoc XRSPlus (Bio-Rad). cells at prometaphase (Schorl and Sedivy, 2007). In addition to
Intensity of chemiluminescence was measured using the ImageJ shortening of the incubation period with nocodazole, nocodazole
software (NIH, USA). concentrations are thought to be critical for the recovery from
nocodazole arrest (Zieve et al., 1980). Previous studies reported
Mitotic index that monopolar spindles were predominant after release from
prometaphase arrest (Sellitto and Kuriyama, 1988) and the cen-
To examine percentages of mitotic cells, HeLa S3 cells were trosomal matrix at the mitotic poles broke into numerous small
treated with 4 mM thymidine for 18 h, washed with PBS, and subse- structures (Moisoi et al., 2002). Furthermore, a combination of
quently cultured in IMDM supplemented with 5% BS and 40 ng/ml thymidine block and nocodazole treatment causes DNA damage in
nocodazole to trap cells at prometaphase. The number of round- human diploid fibroblasts (Wong and Stearns, 2005). These were
shaped mitotic cells was counted every 1 h under a phase-contrast indicative of the toxicity of high concentrations of nocodazole. To
microscope. The mitotic index is defined as the ratio of the number find the lowest concentration of nocodazole enough to arrest cells
of cells in M-phase to the total number of cells. To examine the effect at prometaphase, asynchronous HeLa S3 cells were directly treated
of nocodazole concentrations on mitotic index, asynchronous HeLa with nocodazole at various concentrations for 5 h. Given that the
S3 cells were treated with 0–80 ng/ml nocodazole for 5 h and fixed cell cycle of HeLa S3 cells is around 18∼20 h, the mitotic index
 Y. Matsui et al. / European Journal of Cell Biology 91 (2012) 413–419 415

Furthermore, it is quite difficult to synchronize cells in anaphase

because of the relatively short duration of anaphase. Since non-
muscle myosin II ATPase motor provides the force for furrow
ingression, it was reported that treatment with the myosin II
inhibitor blebbistatin leads to cytokinesis failure through inhibi-
tion of the cleavage furrow ingression (Straight et al., 2003). Even
though the cleavage furrow ingression is completely inhibited,
sister chromatids are segregated normally, which generates bin-
ucleated cells (Martineau et al., 1995). However, we found that
treatment with blebbistatin at a low concentration (50 ␮M) par-
tially inhibited the furrow ingression and the central spindle in
some of the cells was still thickened during a 50-min incubation,
while the cleavage furrow completely ingressed without blebbis-
tatin and a single nucleus was reformed in both daughter cells that
were still connected with a cell–cell bridge containing the midbody
(Fig. 2). Thus, we can assume that treatment with 50 ␮M blebbis-
tatin slows down the cleavage furrow ingression and extends the
duration of anaphase.
Next, to examine whether cell populations were enriched
in anaphase using 50 ␮M blebbistatin, asynchronous cells were
sequentially treated with 4 mM thymidine and 20 ng/ml nocoda-
zole [Fig. 3A, Method-(i)]. Nocodazole-arrested cells were released
for 20 min, then treated with 50 ␮M blebbistatin and fixed at
the times indicated in Fig. 3C-Method-(i). Nocodazole treatment
arrested most of the cells at prometaphase [Fig. 3B, C-Method-
(i), 0 min]. After nocodazole release, prometaphase-arrested cells
rapidly progressed into metaphase and the number of metaphase
cells peaked at 20 min after the release [Fig. 3B, C-Method-(i),
20 min]. It is interesting to note that approximately 40% of the
cells were successfully enriched in anaphase at 40 min after the
Fig. 1. Time course of mitotic index and dose response of nocodazole. (A) HeLa S3
cells were treated with 4 mM thymidine for 18 h, washed with PBS, and subsequently release, and the proportion of telophase cells increased from 40 min
cultured in the presence of 40 ng/ml nocodazole. The number of round-shaped and reached more than 80% at 70 min [Fig. 3B, C-Method-(i)].
mitotic cells was counted under a phase-contrast microscope at the indicated times Western blotting analysis showed that the expression levels of
after release from the thymidine arrest. More than 143 cells were counted at each
cyclin B1, phospho-histone H3 at Ser10, phospho-Aurora kinases
time point. (B) Asynchronous HeLa S3 cells were treated with 0–80 ng/ml nocoda-
zole for 5 h and fixed with 4% paraformaldehyde. The number of mitotic cells was
A/B/C, and Mad2 were down-regulated during the progression
counted under a phase-contrast microscope (n > 138). from 20 min to 70 min (Fig. 4). In sharp contrast, typical sequential
treatment with 4 mM thymidine and 40 ng/ml nocodazole did not
yield apparent enrichment of cells in anaphase [Fig. 3A, C-Method-
was expected to reach up to 25% after a 5-h incubation of asyn- (ii)]. By following the Method-(i), efficient release from nocodazole
chronous cells with nocodazole. Indeed, we found that the mitotic arrest and synchronization during M-phase progression were also
index increased in a dose-dependent manner and reached approx- observed in human breast cancer MCF-7 [Fig. 3D-Method-(i)]
imately 25% upon treatment with 20 ng/ml nocodazole (Fig. 1B). and human colorectal carcinoma HCT116 cells [Fig. 3E-Method-
These results suggest that the concentration of nocodazole can be (i)], although enrichment of metaphase cells and anaphase cells
reduced to 20 ng/ml for arresting most of the cells at prometaphase. were less efficient in HCT116 cells than HeLa S3 cells and

Fig. 2. Effect of a low concentration of blebbistatin on the cleavage furrow ingression. Thymidine-arrested HeLa S3 cells were released for 9 h and then incubated with
nocodazole at 20 ng/ml. After 4-h incubation with nocodazole, round-shaped mitotic cells were collected by mitotic shake-off, washed and released into nocodazole-free
fresh medium for 20 min. Then, cells were cultured in the presence of 0 or 50 ␮M blebbistatin for 50 min, fixed and stained for ␣-tubulin (green) and DNA (red). Magnified
images are shown right. Scale bars, 10 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
416 Y. Matsui et al. / European Journal of Cell Biology 91 (2012) 413–419

Fig. 3. Use of blebbistatin for synchronization during M-phase progression. (A) Schematic depiction of the synchronization methods. Method-(i): HeLa S3 cells treated with
4 mM thymidine for 24 h were released for 9 h and incubated with nocodazole at 20 ng/ml for 4 h. Mitotic cells were collected by mitotic shake-off, washed and resuspended
with drug-free fresh medium. After 20-min release from nocodazole arrest, cells were incubated with blebbistatin and fixed at the indicated times. Method-(ii): Mitotic cells
were collected as in method-(i) except for the nocodazole concentration at 40 ng/ml. Mitotic cells were washed and incubated in the absence of blebbistatin, followed by
fixation at the indicated times. W, washing with PBS. (B) Cells were synchronized as described in Method-(i), fixed at the indicated times and stained for ␣-tubulin (green)
and DNA (red). Representative images of cells are shown. Scale bars, 10 ␮m. (C) [Method-(i)] HeLa S3 cells were synchronized as described in Method-(i), stained as shown
in (B) and examined for mitotic phases. More than 200 cells were examined at each time point. The data are average value ± SDs from three independent experiments.
Representative images of each mitotic phase are shown right. [Method-(ii)] HeLa S3 cell were synchronized as described in Method-(ii), stained as shown in (B) and were
examined for mitotic phases. More than 195 cells were examined at each time point. The data are average value ± SDs from three independent experiments. (D) MCF-7 cells
were synchronized as described in Method-(i) and Method-(ii), stained as shown in (B) and examined for mitotic phases. More than 126 cells were examined at each time
point. The data are average value ± SDs from three independent experiments. In Method-(i), approximately 27% ± 13% cells were binucleated at 70 min after the release
from nocodazole arrest possibly through inhibition of myosin II. These cells were counted as “Telophase”. (E) HCT116 cells were synchronized as described in Method-(i)
and Method-(ii), stained as shown in (B) and examined for mitotic phases. More than 106 cells were examined at each time point. The data are average value from two
independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
 Y. Matsui et al. / European Journal of Cell Biology 91 (2012) 413–419 417

Fig. 3. (Continued)

MCF-7 cells (Fig. 3C–E). Taken together, these results suggest that results, we decided to use HeLa S3 cells in the following experi-
after release from thymidine arrest at G1/S phase, use of low con- ments.
centrations of nocodazole and blebbistatin is highly effective for Src-family tyrosine kinases (SFKs) consist of 8 members and
enrichment of cells in anaphase and telophase. Based on these have roles in cell proliferation, adhesion, migration, and devel-
opment (Brown and Cooper, 1996; Thomas and Brugge, 1997).
Accumulating evidence suggests that SFKs regulate cell division
(Kasahara et al., 2007; Moasser et al., 1999; Ng et al., 2005;
Roche et al., 1995; Tominaga et al., 2000). On mitotic entry, Cdk1-
mediated phosphorylation of SFKs leads to their mitotic activation
(Bagrodia et al., 1991; Chackalaparampil and Shalloway, 1988;
Erpel and Courtneidge, 1995; Kesavan et al., 2002; Kuga et al., 2007;
Morgan et al., 1989; Pathan et al., 1996; Shenoy et al., 1989; Zheng
and Shalloway, 2001). We recently reported that electrophoretic
mobilities of c-Src and c-Yes are retarded in prometaphase in a
Cdk1 activity-dependent manner and the mobility shifts disappear
after mitotic exit, and that the retarded mobility form of c-Yes is
highly activated (Kuga et al., 2007). However, dynamic changes in
electrophoretic mobility of SFKs during M-phase progression are
not yet fully clarified.
To examine dynamic changes in mitotic modification of SFKs,
cells were synchronized as described in Fig. 3A [Method (i)] and sol-
ubilized at 0 min (prometaphase, >80%), 20 min (metaphase, >60%),
40 min (anaphase, >40%), and 70 min (telophase, >80%) after release
from nocodazole arrest. Western blots probed with anti-Yes anti-
body showed that the retarded mobility form of c-Yes was seen
in prometaphase and metaphase and the normal mobility form
of c-Yes was detected not in prometaphase and metaphase but
in telophase (Fig. 5A), consistent with our recent studies (Kuga
et al., 2007). Intriguingly, we found that a new retarded mobility
form of c-Yes was detected in anaphase and slightly in telophase,
which was electrophoretically located between the retarded form
Fig. 4. Biochemical analysis of mitotic markers during M-phase progression. HeLa in prometaphase/metaphase and the normal mobility form in
S3 cells were synchronized in each mitotic phase as described in Method-(i). Total
cell lysates were obtained at 0, 20, 40, and 70 min after release from nocodazole
telophase (Fig. 5A, 2.7% C and 5% C crosslinking gels). The degree
arrest and subjected to Western blotting, probed with anti-phospho-histone H3 of mobility shift of c-Yes in anaphase was moderate compared
(Ser10), anti-histone H3, anti-cyclin B1, anti-phospho-Aurora A/B/C, anti-Aurora A, with that in prometaphase and metaphase, indicating that there
anti-Aurora B, anti-Mad2 and anti-␣-tubulin (loading control) antibodies. The bands is an anaphase-specific modification form of c-Yes. We therefore
detected with anti-phospho-Aurora A/B/C were assigned to phospho-Aurora A,
demonstrate for the first time the three different mobility forms
phospho-Aurora B and phospho-Aurora C based on their molecular weights (Aurora
A, 46 kDa; Aurora B, 39 kDa; Aurora C, 36 kDa). This was confirmed by reprobing of of c-Yes. When we examined the other SFK members for their
the membrane with anti-Aurora A and anti-Aurora B antibodies.
418 Y. Matsui et al. / European Journal of Cell Biology 91 (2012) 413–419

Fig. 5. Dynamics of mitotic regulator proteins during M-phase progression. HeLa S3 cells were synchronized in each mitotic phase as described in Method-(i). Total cell
lysates were obtained at 0 (prometaphase), 20 (metaphase), 40 (anaphase), and 70 min (telophase) after release from nocodazole arrest and subjected to Western blotting,
probed with anti-Yes, anti-Src (GD11), anti-Lyn, anti-Fyn, anti-phospho-pERK1/2, anti-ERK2, anti-cyclin B1, anti-actin (loading control) and anti-␣-tubulin (loading control)
antibodies. In A and B, Western blotting analysis were performed with both 2.7% C and 5% C crosslinking gels. Arrows indicate retarded- and normal-mobility forms of c-Yes
(A) and c-Src (B). In D, the bands detected with anti-phospho-ERK1/2 were assigned to phospho-ERK1 and phospho-ERK2 based on their molecular weights (pERK1, 44 kDa;
pERK2, 42 kDa). This was confirmed by reprobing of the membrane with anti-ERK2 antibody.

electrophoretic mobilities during M-phase progression, retarded These results suggest that activation of ERK1/2 takes place upon
mobility shifts were seen in c-Src from prometaphase to anaphase mitotic entry, supporting the hypothesis that ERK1/2 are activated
whereas those were not in Lyn and Fyn (Fig. 5B, 2.7% C and 5% C in prometaphase/metaphase by SFK-mediated signaling at the
crosslinking gels, C), consistent with our recent results (Kuga et al., plasma membrane and/or Rab11-associated recycling endosomes
2007). These results suggest that phosphorylation and dephospho- and activated ERK1/2 together with activated MEK are subse-
rylation by Cdk1 and a mitotic phosphatase might influence the quently delivered to the midbody via vesicle transport (Kasahara
activity and/or localization of SFKs during the progression from et al., 2007).
prometaphase/metaphase to telophase through anaphase. In conclusion, we developed a novel method for enriching cell
Furthermore, we recently showed that recruitment of activated populations in metaphase, anaphase, and telophase by synchro-
ERK, which is phosphorylated by MEK downstream of SFKs, to nization using low concentrations of blebbistatin and nocodazole.
the midbody plays an important role in completion of abscission The cell populations obtained by our method are suitable for
(Kasahara et al., 2007). To examine the activation levels of ERK1/2 biochemically analyzing dynamic changes in proteins during M-
during M-phase progression, Western blotting analysis was per- phase progression. Our procedure for synchronization is depicted
formed using anti-phospho-ERK antibody. Prolonged activation in Fig. 3A [Method-(i)]. HeLa S3 cells treated with 4 mM thymidine
of ERK1/2 was seen during the progression from prometaphase for 24 h are released for 9 h and incubated with 20 ng/ml nocoda-
to telophase despite a slight decrease in the activation (Fig. 5D). zole for 4 h. Then, mitotic cells are collected by mitotic shake-off,
 Y. Matsui et al. / European Journal of Cell Biology 91 (2012) 413–419 419

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