Aza-BODIPY probe for selective visualization of cyclooxygenase-2 in cancer cells

Article abstract of DOI:10.1039/c9ra01948k

AZB-IMC₂ was developed as a COX-2 specific probe that exhibited a brighter fluorescence signal in cancer cells that overexpress COX-2 compared to normal cells. Oxidative stress agent-treated inflamed cell lines inducing high COX-2 levels revealed an enhanced fluorescence signal. Inhibitory studies showed a markedly reduced fluorescence intensity in cancer cells. The results suggested that AZB-IMC₂ could be developed as a promising molecular tool for imaging guiding during surgery.

Full text of DOI:10.1039/c9ra01948k

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Aza-BODIPY probe for selective visualization of
cyclooxygenase-2 in cancer cells†
Cite this: RSC Adv., 2019, 9, 13372
c
Thitima Pewklang,‡a Kantapat Chansaenpak,‡b Rung-Yi Lai,a Parinya Noisa
a
*
and Anyanee Kamkaew
AZB-IMC2 was developed as a COX-2 specific probe that exhibited a brighter fluorescence signal in cancer
cells that overexpress COX-2 compared to normal cells. Oxidative stress agent-treated inflamed cell lines
inducing high COX-2 levels revealed an enhanced fluorescence signal. Inhibitory studies showed
a markedly reduced fluorescence intensity in cancer cells. The results suggested that AZB-IMC2 could be
developed as a promising molecular tool for imaging guiding during surgery.
Received 14th March 2019
Accepted 26th April 2019
DOI: 10.1039/c9ra01948k
rsc.li/rsc-advances
a quinolinium-based moiety conjugated to IMC using click chem-
istry and the conjugate showed remarkable targeted ability towards
cancer cells over normal cells regarding the COX-2 levels.29
Introduction
Cyclooxygenase (COX), a prostaglandin-endoperoxide synthase,
is a family of the two isoenzymes, COX-1 and COX-2, and plays
an essential role in the formation of prostanoids.1 Among the
isoenzymes, clinical data revealed that COX-2 is overexpressed
in all stages of cancer, from the earliest premalignant phase to
metastasis2 and high levels of COX-2 were found to be upregu-
lated in various types of cancers, such as pancreatic, colon,
gastric, stomach, breast, head/neck carcinoma, or inammatory
lesions.3–11 Therefore, COX-2 has been considered as a prom-
ising target for tumor-specic target-based therapeutics.6,12–18 In
the past decades, some uorescence-based COX-2-targeting
probes have been developed,19–25 however, COX-2-specic
cancer biomarker development is still needed for clinical use.
Indomethacin (IMC), a commercially available nonsteroid anti-
inammatory drug (NSAID), exerts tumor-selective diagnosis and
therapeutic potency by targeting COX-derived prostaglandin (PG)
biosynthesis.26,27 Although IMC is a non-selective COX isoforms
inhibitor (COX-1 and COX-2), it becomes more selective aer
conjugation with uorescent dyes.6,24,28–31 For example, indometh-
acin (IMC) was linked to Nile Blue dye using a hexanediamine linker
as a NIR uorescent imaging probe.30 The probe showed Golgi
localization in cancer cells that overexpressed COX-2. Moreover, this
NIR probe preferentially labeled the tumors in vivo, suggesting
potential tool for imaging guided surgery. Another study employed
In general, dyes that absorb and emit light in NIR region are
ideal to minimize interference from cells autouorescence, which
is a major background noise in cell imaging. However, most
currently used NIR-emitting dyes in bioimaging, particularly cya-
nine-based uorophores, suffer from inherently low chemical and
photostability.32–34 These phenomena signicantly limit the use of
these dyes in long-term optical imaging that requires duration of
light exposure in cellular environment. Borondipyrromethenes
(BODIPYs) and their derivatives, in comparison, contain boron
bridge that introduces rigidity to the system leading to prevention
of trans–cis isomerization and twisting, enhancing uorescent
quantum yield and photostability of the dyes.35–38
According to the bivalent ligand approach, it was found that
duplication pharmacophores leads to an increase in potency of
binding recognition compared to the corresponding mono-
valent ligand.39–44 Therefore, in this study, we reported bivalent
conjugation of indomethacin to an aza-borondipyrromethene
(aza-BODIPY) dye, a class of BODIPYs with NIR emission,45 to
enhance COX-2 selectivity (AZB-IMC2).
Aza-BODIPY dye was linked to two molecules of indomethacin
using azidotriethyleneglycol linker (Scheme 1). AZB-IMC2 has
advantages of optical properties with NIR absorption (lmax ꢀ 700
nm) and emission (lmax ꢀ 730 nm) which can detect cancer cells
by uorescence imaging. NIR uorescent COX-2 inhibitors are
attractive candidates as deep-seated tissue targeted imaging
agents. Such compounds are nonradioactive and stable, thus,
they can be developed conveniently for clinical imaging agents.
aSchool of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon
Ratchasima, Thailand 30000. E-mail: anyanee@sut.ac.th
bNational Nanotechnology Center, National Science and Technology Development
Agency, Thailand Science Park, Pathum Thani, Thailand 12120
cLaboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of
Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima,
Thailand 30000
Results and discussion
Synthesis of AZB-IMC2
† Electronic supplementary information (ESI) available: Detailed synthesis and
characterizations of AZB-IMC2. See DOI: 10.1039/c9ra01948k
‡ These authors contributed equally to this article.
AZB-IMC2 was synthesized according to Scheme 1. Compound 2
was obtained from the reaction of sodium azide with 2-(2-(2-
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Table 1 Photophysical properties of AZB-IMC2
lmax
Dl
(nm)
3 (Mꢁ1 cmꢁ1
)
lemiss (nm)
Ffb
a
Solvent
(nm)
DMSO
704
694
710
710
2.5 ꢂ 10ꢁ4
2.5 ꢂ 10ꢁ4
1.7 ꢂ 10ꢁ4
1.5 ꢂ 10ꢁ4
734
722
725
725
30
28
15
15
0.47
0.80
0.01
0.05
CHCl3
H2Oc
PBSc pH 7.4
a
b
Samples were excited at 670 nm. Relative to Zn-phthalocyanine in
pyridine (Ff ¼ 0.30). c With 3% Tween-80.
Cell viability
Next, cell viability of AZB-IMC2 were tested in different cell lines
including cancer (HeLa, HepG2 and MCF-7) and normal
(Hek293, RAW 264.7 and HFF) cells. The cells were treated with
various concentrations of AZB-IMC2 ranging from 0–50 mM for
24 h before quantifying cell viability. Fig. 2 showed all the cells
remained more than 80% viability at the concentration up to 20
mM while at higher concentration (50 mM) the cell viability
reduced to only 50%. This phenomenon might be due to the
aggregation of the probe at high concentration. However, the
highest concentration we used in the following experiments was
5 mM which all the cells maintained full viability.
Scheme 1 Synthesis of AZB-IMC2.
chloroethoxy)ethoxy)ethanol that give quantitative yield.46 Aer
that, 2 was undergone esterication with indomethacin to give
compound 3. In another part, amino aza-BODIPY 4 47 was amide
coupled with propiolic acid to obtain alkyne substituted aza-
BODIPY 5. Finally, the azide–alkyne Huisgen cycloaddition
between the azide 3 and the alkyne 5 was performed to give AZB-
IMC2 which was used as a COX-2 specic probe for cancer cells
imaging.
Confocal imaging
COX-2-targeting ability of AZB-IMC2 was then investigated in
living cells. We selected 6 different cell lines, cancer cells (HeLa,
HepG2 and MCF-7) known to overexpress COX-2 and normal
cells with lower expression of COX-2 (HEK293, HFF and RAW
264.7).19,24,25,30,48 Aer all the cells exposed to AZB-IMC2,
increased uorescent signals were observed when the time
increased. Moreover, the brighter uorescence was detected in
cancer cells compared to normal cells and the differences were
obviously observed aer 6 h incubation (Fig. 3).
In addition, in cancer cells, the uorescence was found to be
in dose-dependent manner (Fig. 4). We also noticed that 5 mM of
AZB-IMC2 was enough for cell internalization and could be used
as an indicator to distinguish cancer from normal cell lines.
Optical properties
AZB-IMC2 showed strong UV-vis-NIR absorption peaking at
704 nm and emitted bright uorescence peaking at 734 nm in
DMSO (Fig. 1). The optical properties were determined under
various conditions (DMSO, CHCl3, H2O + 3% Tween-80 and
0.01 M PBS pH 7.4 + 3% Tween-80) however, the absorption and
emission maxima did not shi signicantly. In water and PBS
media, Tween-80, the biocompatible surfactant, was added to
increase solubility of the dye (see ESI†). AZB-IMC2 exhibited
high quantum yield in organic solvents, whereas low quantum
yield was observed in aqueous media (Table 1). This might be
the results from hydrophobic nature of the targeting probe
leading to aggregation in aqueous medium. As AZB-IMC2 could
absorb and emit lights in NIR region, this could be benecial
for deep tissue imaging.
Fluorescence images of lipopolysaccharide-induced
inammatory cell lines
To expand an application of AZB-IMC2 to detect intracellular
COX-2 inamed cells, HEK-293, RAW 264.7 and broblast (HFF)
Fig. 1 UV-vis-NIR absorption and fluorescent spectra of AZB-IMC2 Fig. 2 Effect of AZB-IMC2 on cell viability in different cell lines. All cells
(2.9 mM) excited at 670 nm in DMSO.
were treated with AZB-IMC2 at different concentrations for 24 h.
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Fig. 6 Confocal images of COX-2 inhibitory effect. Cancer cells were
incubated with COX-2 specific inhibitors (aceclofenac and celecoxib,
10 mM and 20 mM) for 3 h, then incubated with AZB-IMC2 (5 mM) for
another 3 h.
indomethacin units, the COX-2 blocking experiments were
performed. Cancer cells were incubated with 10 mM and 20 mM
of potent COX-2 specic inhibitors (aceclofenac and cele-
coxib)49,50 for 3 h to down-regulate COX-2 levels before adding
AZB-IMC2. Fig. 6 shows that all cancer cells (HeLa, HepG2 and
MCF-7) incubated with AZB-IMC2 in the absence of COX-2
inhibitors exhibited bright uorescence, whereas, a signicant
reduction in the uorescent intensity was observed when the
cells exposed to COX-2 inhibitors prior to AZB-IMC2 treatment.
Moreover, the uorescence intensity decreased with increasing
amount of celecoxib and aceclofenac, implying that celecoxib
and aceclofenac prevented the labeling of cancer cells by AZB-
IMC2. Therefore, the indomethacin-conjugated probe AZB-IMC2
displayed a clear relationship with COX-2 expression levels.
Fig. 3 Confocal and fluorescence microscopy images of AZB-IMC2 (5
mM) depend on incubation time (1, 3, 6, and 24 h).
Fig. 4 Confocal images of dose-dependent effect of AZB-IMC2 (2.5 and
5.0 mM) in HeLa, HepG2, and MCF-7 cells, which were incubated for 3 h.
Conclusions
In summary, we successfully developed the COX-2 specic
probe (AZB-IMC2) for NIR cancer imaging. AZB-IMC2 contained
two moieties of indomethacin showing strong absorption and
uorescence in the NIR region (above 700 nm). AZB-IMC2 was
not harmful to both cancer (HeLa, HepG2 and MCF-7) and
normal (Hek293, RAW 264.7 and HFF) cells at the concentration
up to 20 mM. Moreover, as shown in confocal images, AZB-IMC2
exhibited high selectivity towards cancer cells where COX-2 is
overexpressed aer at least 3 h incubation. In addition, LPS-
treated inamed cell lines (Hek293, RAW 264.7 and HFF)
showed enhanced uorescence as COX-2 level was upregulated.
On the other hand, inhibition of COX-2 levels in cancer cells
caused signicant decreasing of uorescent signals. These
results conrmed that AZB-IMC2 responded to COX-2 expres-
sion level of the cells, which can be useful in development of
a COX-2-specic cancer biomarker for clinical applications.
cell lines were rst induced with LPS (lipopolysaccharides) to
upregulate COX-2 levels. Aer incubation with LPS for 12 h,
followed by treatment with AZB-IMC2 for 2 h, an enhanced
uorescence was observed in all cell lines (Fig. 5), indicating
that AZB-IMC2 could be used to monitor the oxidative stress in
the living system.
COX-2 inhibition experiment
Moreover, to evaluate the selectivity towards cancer cells over
normal cells via COX-2 mediated endocytosis upon binding to
Experimental
Materials and instruments
Fig. 5 Confocal images of LPS-induced inflammation cell lines. HFF,
HEK-293 and RAW 264.7 were incubated with LPS (1 mg mLꢁ1) for 12 h
before exposing to AZB-IMC2 (5 mM) for 2 h. The fluorescent signals
enhanced in the LPS-induced oxidative stress cells.
All glassware was oven-dried prior to use. All the reagents have
been purchased from the commercially sources (Sigma Aldrich,
TCI, Carlo Erba, Acros, Merck) and used without further
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purication. Column chromatography purications were per- 7.74–7.68 (m, 1H), 7.50 (s, 1H), 7.39–7.37 (m, 1H), 6.96 (d, J ¼
formed using silica gel for chromatography (Carlo Erba) as 8.7 Hz, 2H), 6.64 (s, 1H), 3.86 (s, 3H), 2.97 (s, 1H). 13C NMR (500
a stationary phase. Analytical thin layer chromatography (TLC) MHz, CDCl3): d ¼ 162.3, 161.2, 150.0, 145.4, 142.2, 137.6, 133.5,
was performed on TLC Silica gel 60 F254 (Merck) and visualized 132.0, 131.4, 129.5, 126.1, 121.2, 119.4, 114.5, 114.1, 75.4, 74.8,
1
with UV cabinet. H NMR and 13C NMR spectra were recorded 55.7 ppm. MS (MALDI-TOF) m/z: the calculated value (calcd) for
on Bruker-500 MHz spectrometer at room temperature. Chem- C40H28BF2N5NaO4 ([M + Na]+): 714.21, C40H28BF2KN5O4 ([M +
ical shis of 1H NMR spectra were recorded and reported K]+): 730.18, found 714.27, 730.23.
in ppm from the solvent resonance (CDCl3 at 7.24 ppm). Data
Synthesis of (((((((((5,5-diuoro-3,7-bis(4-methoxyphenyl)-5H-
are reported as follows: chemical shi, multiplicity (s ¼ singlet, 4l4,5l4-dipyrrolo[1,2-c:20,10-f][1,3,5,2]triazaborinine-1,9-diyl)bis(3,1-
d ¼ doublet, t ¼ triplet, m ¼ multiplet), coupling constants, and phenylene))bis(azanediyl))bis(carbonyl))bis(1H-1,2,3-triazole-4,1-
number of protons. 13C NMR spectra were also recorded in ppm diyl))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(-
from the solvent resonance (CDCl3 at 77.23 ppm). MS were ethane-2,1-diyl)-bis(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
measured under ESI and MALDI conditions.
indol-3-yl)acetate) (AZB-IMC2). 5 (54 mg, 0.078 mmol) and 3
(0.1178 g, 0.2287 mmol, 3 eq.) were dissolved in dimethyl sulf-
oxide (DMSO, 5 mL). To the reaction mixture was added sodium
ascorbate (0.2 mmol, 400 mL of freshly prepared 0.5 M solution in
Synthetic procedures
Synthesis of 2-(2-(2-azidoethoxy)ethoxy)ethyl-2-(1-benzoyl-5- DI water) and copper(II) sulfate pentahydrate (0.12 mmol, 240 mL
methoxy-2-methyl-1H-indol-3-yl)acetate (3). To solution of 2 of freshly prepared 0.5 M solution in DI water), respectively. The
(0.2498 g, 1.474 mmol) in dry dichloromethane (DCM, 5 mL) was resulting mixture was stirred vigorously at room temperature for
added indomethacin (0.4904 g, 1.371 mmol). The reaction 24 h. Aer that, DCM (20 mL) was added to the mixture and the
mixture was cooled to 0 ꢃC. Then, N,N0-dicyclohexylcarbodiimide organic layer was washed with DI water (3 ꢂ 20 mL). The DCM
(DCC, 0.4400 g, 2.132 mmol) and 4-dimethylaminopyridine layer was then dried over anhydrous Na2SO4 and the solvent was
(DMAP, 48.2 mg, 0.394 mmol) were added into the mixture at removed under reduced pressure. The obtained residue was
0
ꢃC. The reaction mixture was allowed to warm to room puried by silica chromatography eluting with hexane : EtOAc
temperature and stirred for 24 h. Aer that, the reaction was (1 : 1 to 1 : 3), followed by DCM : MeOH (95 : 5) to yield 20 mg
1
ltered through Celite® with DCM as eluent. The organic layer (15%) of AZB-IMC2 as a dark green solid. H NMR (500 MHz,
was washed with DI water (3 ꢂ 20 mL) and brine (20 mL), and CDCl3): d ¼ 8.97 (s, 1H), 8.35 (s, 1H), 8.29 (s, 1H), 8.08 (d, J ¼
dried over anhydrous Na2SO4. The solvent was then removed 8.8 Hz, 2H), 7.84 (d, J ¼ 7.8 Hz, 1H), 7.68 (d, J ¼ 7.9 Hz, 2H), 7.59
under reduced pressure. The obtained residue was puried by (s, 1H), 7.57 (s, 1H), 7.45–7.40 (m, 1H), 7.07 (s, 1H), 6.98 (d, J ¼
silica chromatography eluting with DCM : MeOH (100 : 0 to 8.8 Hz, 2H), 6.92 (s, 1H), 6.81 (d, J ¼ 9.0 Hz, 2H), 6.60 (d, J ¼
90 : 10) to yield 0.3062 g (43%) of 3 as a yellow sticky oil. 1H NMR 8.9 Hz, 1H), 4.58 (s, 2H), 4.26 (t, J ¼ 4.6 Hz, 2H), 4.05–4.04 (m,
(500 MHz, CDCl3): d ¼ 7.62 (d, J ¼ 8.5 Hz, 2H), 7.43 (d, J ¼ 8.4 Hz, 2H), 3.87 (s, 3H), 3.81 (m, 2H), 3.76 (s, 3H), 3.66–3.63 (m, 2H),
2H), 6.94 (d, J ¼ 2.4 Hz, 1H), 6.85 (d, J ¼ 9.0 Hz, 1H), 6.64–6.62 (m, 3.54–3.52 (m, 2H), 3.45 (t, J ¼ 3.7 Hz, 2H), 2.31 (s, 3H). 13C NMR
1H), 4.24 (t, J ¼ 9.5 Hz, 2H), 3.80 (s, 3H), 3.66 (s, 2H), 3.60 (t, J ¼ (500 MHz, CDCl3): d ¼ 171.2, 168.6, 162.3, 158.5, 158.4, 157.1,
10.1 Hz, 2H), 3.56 (s, 4H), 3.32 (t, J ¼ 10.0 Hz, 2H), 2.34 (s, 3H), 156.3, 143.6, 139.6, 138.2, 136.4, 134.2, 133.6, 132.1, 131.5, 131.0,
2.19 (s, 2H). 13C NMR (500 MHz, CDCl3): d ¼ 170.9, 168.3, 156.1, 129.6, 129.4, 127.4, 125.8, 124.5, 121.1, 120.7, 119.4, 115.2, 114.6,
139.3, 136.1, 134.0, 131.2, 131.2, 129.2, 115.0, 112.6, 111.7, 101.5, 112.8, 112.0, 111.8, 101.9, 70.9, 70.8, 69.5, 64.4, 56.0, 34.3, 34.3,
70.7, 70.6, 70.1, 69.2, 64.2, 55.8, 50.7, 30.3, 13.5 ppm. MS (ESI+) m/ 30.5, 30.0, 13.7 ppm. MS (MALDI-TOF) m/z: the calculated value
z: the calculated value (calcd) for ([M + Na]+): 537.15, found 537.15. (calcd) for C90H83BCl2FN13NaO16 ([M ꢁ F + H]+): 1701.55, C90-
Synthesis of N,N0-((5,5-diuoro-3,7-bis(4-methoxyphenyl)- H82BCl2F2N13NaO16 ([M + Na]+): 1742.53, C90H82BCl2F2KN13O16
5H-4l4,5l4-dipyrrolo[1,2-c:20,10-f][1,3,5,2]triazaborinine-1,9-diyl) ([M + K]+): 1758.51, found 1701.83, 1742.87, 1758.84.
bis(3,1-phenylene))dipropiolamide (5). To solution of 4 (145 mg,
0.247 mmol) in dry dichloromethane (DCM, 5 mL) was added
propiolic acid (0.06 mL, 1 mmol, 8.6 eq.). The reaction mixture
was then cooled to 0 ꢃC. 1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC, 200 mg, 1.04 mmol) and 4-dimethylamino-
pyridine (DMAP, 30 mg, 0.24 mmol) were added into the mixture
at 0 ꢃC. The reaction mixture was allowed to warm to room
temperature and stirred for 24 h. Aer that, the reaction was
ltered through Celite® with DCM as eluent. The organic layer
was washed with HCl (0.2 N, 1 ꢂ 20 mL), NaOH (0.2 N, 2 ꢂ 20
mL) and brine (20 mL), respectively. The organic layer was then
dried over anhydrous Na2SO4 and the solvent was removed under
reduced pressure. The obtained residue was puried by silica
chromatography eluting with hexane : EtOAc (1 : 1 to 1 : 3) to
UV/vis and uorescence spectroscopic methods
All the UV/vis absorption spectra and uorescence were recor-
ded on UV-vis Spectrophotometer (Agilent Technologies Cary
300) and Spectrouorometer (PerkinElmer LS55), respectively.
In both experiments, stock solutions (350 mM) of AZB-IMC2
probe was prepared in DMSO. 10 mM solutions of probes in
chloroform, DMSO, DI water, and 0.01 M PBS buffer (pH 7.4)
with 3% DMSO were prepared as working solutions. For uo-
rescence experiments, the emission spectra were recorded at
excitation wavelength of 670 nm.
Cell culture
yield 69 mg (40%) of 5 as a dark green solid. 1H NMR (500 MHz, Human cervical cancer (HeLa), human hepatoma cell cancer
CDCl3): d ¼ 8.50 (s, 1H), 8.03–8.01 (m, 2H), 7.90 (d J ¼ 8.7 Hz, 1H), (HepG2), Michigan Cancer Foundation-7 (MCF-7), human
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embryonic kidney 293 (HEK-293), Human Foreskin Fibroblast another 3 h. Thereaer, the cells were washed with 0.01 M PBS
(HFF), and RAW264.7 cell lines were cultured on 75 cm3 culture three times and treated with 0.5 mg mLꢁ1 of Hoechst 33342
asks in Dulbecco's Modied Eagle's Media (Hyclone) supple- containing media before visualization by Laser Scanning
mented with 10% fetal bovine serum (Gibco) and 1% penicillin– Confocal Microscope (Nikon A1Rsi).
ꢃ
streptomycin (Corning). All the cells were cultured at 37 C in
a humidied 95% air, 5% CO2 atmosphere.
Conflicts of interest
There are no conicts to declare.
Cell viability assay
The cells were seeded on 96-well plate approximately 7 ꢂ 103
cells per well and incubated in completed media for 24 h.
Thereaer, the cells were treated with 0, 0.5, 5, 10, 20, 50 mM of
AZB-IMC2 and continued culturing for 24 h. Aer incubation,
the cells were washed with 0.01 M PBS (3 times) and treated with
20 mL of methylthiazolyldiphenyl-tetrazolium bromide (MTT
reagent, 0.5 mg mLꢁ1, Sigma-Aldrich) for 2 h. Aer media
removal, DMSO was added to dissolve formazan product. The
cell's viabilities were detected through UV-vis absorption of
formazan at wavelength 560 nm.
Acknowledgements
This research was supported by SUT Research and Development
Fund.
Notes and references
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Time dependent internalization
HeLa, HepG2, MCF-7, HEK-293, HFF and RAW 294.7 cells,
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coverglass (LabTek, Nunc) and incubated in completed media
for 24 h. Aer that, the cells were treated with 5 mM of AZB-IMC2
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Dose dependent internalization
HeLa, HepG2, MCF-7 cells, approximately 1 ꢂ 104 cells, were
seeded on 8-well chambered coverglass and incubated in
completed media for 24 h. Aer that, the cells were treated with
2.5 and 5.0 mM of AZB-IMC2 probe for 3 h. Aer incubation, the
cells were washed with 0.01 M PBS (3 times) and treated with 0.5
mg mLꢁ1 of Hoechst 33342 containing media. The cells were
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