Thiol-inducible direct fluorescence monitoring of drug release

Article abstract of DOI:10.1039/c2ob26680f

A new bifunctional compound NCC, which undergoes thiol-mediated disulfide cleavage after cell entry, produces a red-shifted fluorescent emission in the cytosol and releases free active DNA alkylating agent CLB into the nucleus, and finally leads to DNA damage and cell death.

Full text of DOI:10.1039/c2ob26680f

Organic &
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Thiol-inducible direct fluorescence monitoring of drug
release†
Cite this: Org. Biomol. Chem., 2013, 11,
580
Jun Wu,‡a Rong Huang,‡a Changcheng Wang,a Wenting Liu,a Jiaqi Wang,a
Xiaocheng Weng,a Tian Tiana and Xiang Zhou*a,b
Received 26th August 2012,
Accepted 2nd November 2012
A new bifunctional compound NCC, which undergoes thiol-mediated disulfide cleavage after cell entry,
produces a red-shifted fluorescent emission in the cytosol and releases free active DNA alkylating agent
CLB into the nucleus, and finally leads to DNA damage and cell death.
DOI: 10.1039/c2ob26680f
www.rsc.org/obc
described in the Experiment section. CLB is an alkylating
agent that is used to treat chronic lymphocytic leukemia
Introduction
Prodrugs are inactive compounds prior to administration. through alkylation of the nuclear genome.20–23 Additionally,
While upon exposure to specific physiological conditions, the linkage disulfide bond is supposed to undergo cleavage via
such as acidic, reducing and overexpressed enzymatic milieus, reaction with intracellular abundant thiols, such as gluta-
24–26
they are triggered to degrade or metabolize into an active thione or cysteine.
On the basis of disulfide cleavage, the
therapeutic.1–4 Novel chemotherapeutic drugs that can report fluorophore naphthalimide is released and leads to a distinc-
on its localization and activation in cancer cells are being tive ratiometric emission change. 4-Amino-1,8-naphthalimide
extensively investigated in the cancer treatment field.5–8 is regarded as a useful fluorescent reporter group due to
Examples for selective tumor targeting include folate, cyclic its unique ICT structure, large Stokes’ shift, long emission
peptides and galactose modified cytotoxic drugs (campto- wavelength and high quantum yield.27–30 Our approach is illus-
thecin and taxol) and prodrugs activated by hypoxia trated in Scheme 2. As detailed below, NCC will become fluore-
(tirapazamine).9–15 However, it is difficult to establish precise scent activated in the cytosol upon cell entry that coincides
delivery of a pharmaceutically activatable prodrug to cancer with drug release into the nuclei, resulting in cell apoptosis.
cells.16,17 Toward this end, fluorescent probes that can provide
a ready read-out under the typical conditions of cellular analy-
Optical property studies
sis are of particular interest. It would be desirable to develop a
To demonstrate that the cleavage of the disulfide bond could
drug release system that contains both an active drug for
be effected by treatment with thiols, DTT (1,4-dithiothreitol)
efficacy and a fluorophore for monitoring.18,19
was chosen to be used in vitro for the reaction with NCC, in
consideration of its better liposolubility among commercially
Results and discussion
Herein, we design a new compound naphthalimide–CLB con-
jugate (NCC) which is composed of a potent anticancer drug
chlorambucil (CLB), a disulfide linker and a fluorescent
naphthalimide moiety. NCC was prepared using a mature
route with a satisfactory yield (Scheme 1), that is completely
aCollege of Chemistry and Molecular Sciences, Key Laboratory of Biomedical
Polymers of Ministry of Education, State Key Laboratory of Virology, Wuhan
University, Hubei, Wuhan, 430072, P. R. China. E-mail: xzhou@whu.edu.cn;
Fax: +86-27-87336380; Tel: +86-27-61056559
bState Key Laboratory of Natural and Biomimetic Drugs, Beijing University,
P. R. China
†Electronic supplementary information (ESI)
10.1039/c2ob26680f
available. See DOI:
Scheme 1 Synthesis of NCC. (a) EtOH–butylamine; (b) Pd/C, H2; (c) 2,2’-dithio-
‡These authors contributed equally to this work.
diethanol, tri-phosgene, DIPEA; (d) CLB, DCC, DMAP.
580 | Org. Biomol. Chem., 2013, 11, 580–585
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Fig. 2 (a) Fluorescence responses of NCC (10 μM) toward other nonthiol
amino acids (5 mM respectively), including Val, Tyr, Thr, Ser, Pro, Phe, Met, Leu,
Ile, His, Gly, Glu, Gln, Asp, Asn, Arg, Ala, Trp (white bars) and DTT (5 mM) in the
presence of either nonthiol amino acid (black bars). (b) Fluorescence responses
of NCC (10 μM) toward biologically relevant metal ions (Zn2+, Na+, Mg2+, K+,
Fe3+, Cu2+ and Ca2+, 1 mM respectively). Bars represent fluorescence intensity
ratio F533/F475. Each spectrum was acquired 1 h after exposure to the analytes at
37 °C. Excitation wavelength = 420 nm.
Scheme 2 Schematic representation of intracellular thiol mediated disulfide
cleavage to induce fluorescent change and drug release.
metal ions (Fig. 2a and b). Moreover, the effects of the above-
mentioned thiol-free analytes and metal ions on DTT inducing
cleavage were excluded, as the results show that NCC possesses
high selectivity toward DTT when present with these inter-
ferents simultaneously.
Efforts were made to investigate the time-dependent fluor-
escent variation. In Fig. S1,† the emission intensity at 475 nm
was seen to increase initially in the first two minutes, then
decrease gradually. Meanwhile, the emission at 533 nm was
markedly enhanced within 15 min, and then reached a
plateau. Furthermore, the pH dependence of the DTT-induced
emission change was assessed (Fig. S2†). It shows that NCC
provides a stable low background signal within a pH range of
2–10. On the other hand, its response ability toward DTT is
invariable within the biologically relevant pH range (7–9).
Therefore, NCC can readily react with the cellular thiols
without pH effects.
Fig. 1 (a) Absorption change and (b) fluorescence responses of NCC (10 μM)
toward increasing concentrations of DTT (final concentration: 0, 0.01, 0.05, 0.1,
0.5, 1, 2, 4, 5, 6, 8, 10 mM) in sodium phosphate buffer (10 mM, pH = 7.5).
Spectra were acquired 1 h after exposure to DTT at 37 °C. Excitation wavelength
= 420 nm. (c) Fluorescence photographs of NCC in the absence (1) and presence
(2) of DTT. (d) Fluorescence responses of NCC (10 μM) toward thiol-containing
amino acid cysteine (5 mM) after 1 h incubation at 37 °C.
Proposed mechanism of thiol-inducing cleavage
The mechanisms by which the thiol-inducing reaction are pos-
tulated to occur are illustrated in Scheme 3. Complementary
HPLC analysis of the DTT-treated NCC solution demonstrated
the generation of the final fluorescent product naphthalimide.
available thiols. The absorption and fluorescent spectra
changes were monitored upon the addition of DTT under
physiological conditions (phosphate buffer, pH 7.5, 37 °C). As
shown in Fig. 1a and b, with the titration of DTT
(0.01–10 mM), the broad absorption and emission peak cen-
tered in 370 and 475 nm, characteristic of NCC, gradually
underwent a red-shift to 430 and 533 nm respectively, which
corresponded to the product 4-aminonaphthalimide deriva-
tive. The ratio of fluorescence intensities (F533/F475) (λex
=
420 nm) changed from 0.4 to 35 (R = 87.5 fold). In this case, an
easy-to-discern fluorescence change was visualized (Fig. 1c).
The quantum yield of NCC (QY = 0.25) and naphthalimide
derivative (QY = 0.10) were also determined by reference to
known Quinine Sulfate. Similarly, we detected exactly the same
fluorescent response of NCC toward cysteine, a thiol-contain-
ing amino acid which is essential in GSH. However, almost no
appreciable fluorescent changes were observed upon exposure
Scheme 3 Proposed mechanism corresponding to the reaction of NCC with
to nonthiol natural amino acids and biologically essential thiol.
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Fig. 3 The HPLC analysis of NCC without treatment of DTT (top); with DTT
treatment for 2 h at 37 °C (middle); fluorescent naphthalimide only (bottom).
The retention time at 8.9 min corresponds to NCC while the retention time at
5.6 min corresponds to fluorescent product naphthalimide. The signals were
monitored at 503 nm under the irradiation at 382 nm.
Fig. 5 Confocal fluorescence images of thiol-blocking experiment. HeLa cells
incubated with NCC (10 μM) for 1 h (a–c), HeLa cells incubated with NCC
(10 μM) for 1 h after pretreatment with 2.5 mM NEM for 12 h (d–f). (a, d) blue
channel at 450 35 nm, (b, e) green channel at 515 30 nm, (c, f ) bright-field
image. Incubation was performed at 37 °C under a humidified atmosphere con-
taining 5% CO2. Wavelength of excitation laser is 405 nm.
Fig. 4 Agarose gel of plasmid DNA (pBR322) incubated at 37 °C for 12 h with
NCC in the presence of 10 mM GSH. The concentrations of NCC are 50, 100,
250, 500 μM respectively. The first lane is control DNA and the last lane is DNA
marker.
Fig. 6 Alkaline comet assay for HeLa cells treated with NCC (10, 20 and 40 μM)
for 48 h. Nuclear DNA was stained by PI (propidium iodide, a red fluorescent
dye).
As shown in Fig. 3, the peak at the retention time of 8.9 min,
which corresponded to NCC, disappeared after incubation
with DTT. Instead, a new peak with an identical retention time
to naphthalimide (t = 5.6 min) was observed. All these results
confirm that DTT triggers the disulfide cleavage of NCC and
produces a unstable thiol intermediate, which consequently
Intracellular imaging
To determine the fluorescence change of NCC in living cells,
confocal microscopy was performed. HeLa cells incubated
with 10 μM NCC for 1 h displayed intense intracellular fluore-
scence at both blue and green channels (Fig. 5a–c). Further to
confirm such emission resulted from the disulfide cleavage
reaction, a thiol-blocking experiment was conducted. HeLa
cells were pretreated with 2.5 mM NEM (N-ethylmaleimide) for
12 h to downregulate biological thiols and then incubated
with NCC for another 1 h. As expected, the fluorescence inten-
sity at the green channel significantly decreased (Fig. 5d–f). All
the results suggest that thiol-induced disulfide cleavage of
NCC occurs in the cytosol and releases the drug CLB simul-
taneously, which presumably diffuses into the nucleus to func-
tion as DNA alkylating agent.
undergoes
a rapid intramolecular cyclization to releases
naphthalimide for fluorescent signal activation.
DNA alkylation in vitro
As CLB is a DNA alkylating agent, the DNA interacting ability
of NCC upon exposure to GSH was evaluated by denatured
alkaline agarose gel electrophoresis (Fig. 4). After incubation
for 12 h with 10 mM GSH at 37 °C, NCC produced nearly
100% DNA alkylation at a concentration of 500 μM. The non-
reacted supercoiled plasmid DNA (pBR322), which was present
in lower bands, exhibited a dose-dependent decrease in
DNA alkylation in cell systems
accordance with the formation of alkylated DNA in higher The anticancer activity of NCC was evaluated by MTT assay
bands. Hence, NCC is confirmed to trigger DNA damage (Fig. S3†). When HeLa cells were treated with NCC (10 nM–
in vitro. Meanwhile, the well-studied alkylating mechanism of 500 μM) for 72 h, a distinct dose-dependent decrease in cell
CLB is presented in Scheme S1.† The lone pair developed on viability was observed with a IC50 value of 28.8 0.8 μM. To
CLB can form a highly electrophilic intermediate aziridinium explain the origin of cytotoxicity, an alkaline comet assay
ring by intramolecular displacement of the chloride by the related to DNA damage was visually performed and is shown
amine nitrogen. This electrophilic intermediate greatly facili- in Fig. 6. Stained by nuclear red dye PI, the extent of cellular
tates the DNA alkylation and cross-linking formation.
DNA damage increased with increasing concentration of NCC
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release drugs specifically at the diseased site in a more intui-
tive and controllable manner.
Experimental section
Synthesis of compound 3
To a mixture of compound 2 (486.4 mg, 2 mmol) in 50 mL
EtOH was added butylamine (204.8 mg, 2.8 mmol), the result-
ing solution was stirred at reflux temperature for 12 h. After
cooling to room temperature, the solvent and excess butyl-
amine were removed under reduced pressure and the residue
was subjected to flash column chromatography with ethyl
acetate–cyclohexane (1 : 12, v/v) as eluent to give the desired
product as a light yellow solid (416 mg, 70%). 1H NMR
(300 MHz, CDCl3): δ 8.76 (d, J = 8.1 Hz, 1 H), 8.67–8.60 (m,
2 H), 8.29 (d, J = 7.8 Hz, 1 H), 7.91 (t, J = 7.8 Hz, 1 H), 4.12 (t,
J = 6.9 Hz, 2 H), 1.65–1.62 (m, 2 H), 1.39–1.37 (m, 2 H), 0.91 (t,
J = 6.9 Hz, 3 H); 13C NMR (300 MHz, CDCl3): δ 163.5, 162.7,
149.7, 132.6, 130.2, 129.9, 129.4, 127.3, 124.1, 123.3, 40.8, 30.3,
Fig. 7 γ-H2AX immunofluorescence in HeLa cells after incubation without and
with 20, 40 μM NCC for 48 h. The white dotted circles indicate γ-H2AX foci (red)
and the Hoechst 33 258 dye (blue) localizes the nuclear DNA. Images (a, d, g)
were obtained from blue channel at 450 35 nm with excitation wavelengths
of 405 nm, images (b, e, h) were obtained from red channel at 605 35 nm
with excitation wavelengths of 543 nm, and images (c, f, i) are merged photo- 20.5, 14.0 ppm; ESI-MS found m/z = 298.3 [M]+.
graphs of the two channels. Scale bar, 10 μm.
Synthesis of compound 4
This compound was synthesized by hydrogenating 3 (400 mg,
(10–40 μM), resulting in random DNA strand breaks and short
DNA fragment migrations which are observed as comet tails.
At a dose of 40 μM, NCC displayed an obvious comet image,
indicating that it reached target DNA inside the cell and
produced DNA damage as well.
As DNA interstrand cross-links can completely block repli-
cation and transcription and may also generate double stand
breaks (DSBs) during its repair, γ-H2AX immuno-fluorescence
microscopy is a well-accepted and sensitive method to detect
DSBs. After exposure of cells to DSB-inducing agents, the
histone protein H2AX is rapidly phosphorylated on Ser139 as a
signal of DNA damage response.31,32 In Fig. 7, it is noteworthy
that 40 μM NCC triggered prominent red γ-H2AX foci in the
HeLa cell nucleus. In contrast, the control cells did not show
noticeable DNA damage. Therefore, these results confirm that
the thiol-activated cross-linking of NCC induces cytotoxic DNA
damage.
1.34 mmol) in MeOH–CHCl3 (1 : 1) at 30 °C under hydrogen
atmosphere in the presence of 10% Pd/C catalyst for 24 h. The
reaction mixture was filtered, and washed with MeOH and
CHCl3. The filtrate was evaporated under reduced pressure to
1
yield the product as a orange solid (346.6 mg, 95%). H NMR
(300 MHz, CDCl3): δ 8.58 (d, J = 7.8 Hz, 1 H), 8.40 (d, J =
6.0 Hz, 1 H), 8.09 (d, J = 8.1 Hz, 1 H), 7.64 (t, J = 6.0 Hz, 1 H),
6.88 (d, J = 7.5 Hz, 1 H), 4.93 (s, 2 H), 4.14 (t, J = 5.7 Hz, 3 H),
1.71–1.68 (m, 2 H), 1.44–1.41 (m, 2 H), 0.95 (t, J = 5.7 Hz, 3 H);
13C NMR (300 MHz, CDCl3): δ 164.4, 163.5, 153.4, 134.6, 131.6,
130.3, 130.0, 124.6, 122.4, 120.0, 108.8, 108.2, 39.7, 30.5, 20.5,
14.4; ESI-MS found m/z = 267.7 [M − H]−.
Synthesis of compound 5
To a mixture of 4 (0.2 g, 0.74 mmol) and tri-phosgene (0.4 g,
1.4 mmol) in 50 mL of dichloromethane was added DIPEA
(0.5 mL, 2.5 mmol) dropwise at 0 °C. The resulting solution
was stirred for 1 h at room temperature. And then 2,2′-dithio-
diethanol (0.6 g, 3.5 mmol) in CH2Cl2 (5 mL) was added to the
solution and the reaction mixture was stirred for 24 h at room
temperature. The solvent was evaporated off, at which point
Conclusion
In summary, we have developed a multicomponent synthetic CHCl3 (100 mL) and water (100 mL) were added, and the
strategy which allows for fluorescence-activatable monitoring organic layer was collected. The CHCl3 layer was dried over
of the release of an anticancer drug. NCC takes advantage of anhydrous NaSO4. After removal of the solvents, the crude
biologically abundant thiols to trigger disulfide bond cleavage, product was purified over silica gel using ethyl acetate–
with subsequent release of naphthalimide moiety as a ratio- hexanes (v/v, 1 : 3) as the eluent to yield 5 as a yellow solid
1
metric fluorescent reporter and CLB as a DNA alkylating agent (169 mg, 51%). H NMR (300 MHz, CDCl3): 8.59 (m, 2 H), 8.34
to induce DNA damage. This approach can provide both thera- (d, 1 H, J = 8.2 Hz), 8.22 (d, 1 H, J = 8.5 Hz), 7.73 (br, 1 H), 7.27
peutic effect evaluation and drug delivery imaging infor- (t, 1 H, J = 8.0 Hz), 4.56 (t, 2 H, J = 6.3 Hz), 4.14 (d, 2 H, J = 7.5
mation, ensuring improved understanding of cellular uptake Hz), 3.94 (t, 2 H, J = 5.6 Hz), 3.07 (t, 2 H, J = 6.3 Hz), 2.96 (t,
and release mechanisms of drugs. Additional exploration and 2 H, J = 5.8 Hz), 2.24 (br, 1 H), 1.69–1.60 (m, 2 H), 1.58–1.43
modification of the compound NCC could lead to the ability to (m, 2 H), 0.97 (t, 3 H, J = 7.3 Hz). 13C NMR (CDCl3, 300 MHz):
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164.1, 163.6, 152.9, 138.8, 132.4, 131.2, 128.8, 126.6, 126.1, Cell survival assay
123.3, 123.0, 117.9, 116.9, 63.8, 60.5, 41.5, 40.2, 37.4, 30.2,
Cells were harvested using trypsin and washed with PBS. The
cells were added into a 96-well plate with 5000 cells per well
and incubated overnight for attachment at 37 °C in a 5% CO2
incubator. The following day, cells were treated with the com-
pounds (10 nM–500 μM) for 72 h. Control cells (without any
compound) were treated under the exact same conditions.
After the incubation period, cell survival was evaluated using
an MTT assay: 10 μL of a freshly prepared solution of MTT
(5 mg mL−1 in PBS) was added to each well, and after 4 h incu-
bation, the medium was removed; upon addition of 100 μL of
DMSO to each well, the optical density values were detected at
492 nm. Cytotoxicity data were expressed as IC50 values. Data
were expressed as mean values of three individual experiments
conducted in triplicate.
20.4, 13.8 ppm. ESI-MS found 449.1 [M + H+].
Synthesis of compound 1 (NCC)
N,N′-Dicyclohexylcarbodiimide (DCC: 50 mg, 0.24 mmol) was
added to a stirred solution of 5 (100 mg, 0.22 mmol), chloram-
bucil (CLB: 67 mg, 0.22 mmol), and 4-dimethyl-aminopyridine
(DMAP: 5 mg, 0.02 mmol) in CH2Cl2 (5 mL) at 0 °C. The solu-
tion was stirred for 24 h at room temperature. After removal
of dicyclohexylurea by filtration, the filtrate was successively
washed with water (3 × 10 mL) and a saturated solution of
NaCl (5 mL). The solution was dried over NaSO4 and the
solvent removed in vacuo. The crude product was purified over
silica gel using ethyl acetate–hexanes (v/v, 1 : 10) as the eluent
1
to yield 5 as a yellow solid (67 mg, 42%). H NMR (300 MHz,
Confocal imaging of thiol-blocking experiment
CDCl3): 8.61 (m, 2H), 8.35 (d, 1H, J = 8.2 Hz), 7.96 (d, 1H, J =
8.5 Hz), 7.70–6.98 (m, 2H), 6.60–6.57 (m, 2H), 4.54 (t, 2H, J =
6.3 Hz), 4.41 (d, 2H, J = 7.5 Hz), 4.17 (t, 2H, J = 6.3 Hz), 4.02
(d, 2H, J = 7.5 Hz), 3.67 (d, 4H, J = 5.6 Hz), 3.61 (d, 4H, J =
5.6 Hz), 3.04 (t, 2H, J = 6.3 Hz), 2.96 (t, 2H, J = 5.8 Hz), 2.46 (br,
1H), 1.87 (d, 2H, J = 7.5 Hz), 1.43 (m, 2H), 1.25 (m, 2H), 0.97 (t,
3H, J = 7.3 Hz). 13C NMR (CDCl3, 300 MHz): 173.7, 163.6,
153.0, 144.3, 132.3, 131.1, 129.5, 126.4, 123.3, 117.0, 112.1,
67.29, 62.9, 62.4, 53.5, 40.4, 40.1, 37.6, 36.5, 33.8, 33.5,
32.2, 30.1, 26.6, 20.3, 18.6, 13.7, 13.5 ppm. HRMS (ESI): m/z:
calcd for C35H41Cl2N3O6S2 + Na: 756.1712; found: 756.1710
[M+ + Na]; melting range: 175–177 °C.
105 Cells seeded on two 35 mm glass-bottomed dishes (Nest)
were washed with 1 mL PBS. One dish of cells was incubated
with NADH-CLB (10 μM) for 1 h; the other dish of cells was
incubated with NADH-CLB (10 μM) for 1 h after pretreatment
with 2.5 mM NEM for 12 h, and then both of them
were mounted on the microscope stage. The light source was a
white-light laser. The excitation wavelength was 405 nm. Blue
channel at 450 35 nm and green channel at 515 30 nm.
Alkaline comet assay
Cells were plated (4 × 104 cells per well) onto six-well plates to
a final volume of 2 mL and allowed an overnight period for
attachment. Next, each dilution of t NADH-CLB (10–40 μM)
Optical properties study
Fluorescent emission spectra were collected from 460–650 nm was added per well, and the mixtures were incubated at 37 °C
on PerkinElmer LS 55 with an excitation wavelength of for 48 h. After 48 h of incubation, the medium was removed
380 nm, the excitation and emission slit widths were 15 and and replaced with HBSS solution, after which the cells were
9 nm. Quartz cuvettes with 2 mL volume used for emission scraped off. A total of 100 μL of cells was suspended in 700 μL
measurements. UV-Vis absorption spectra were collected on a of 1% low-melting-point agarose and precoated with 1%
SHIMADZU UV-2550 from 350–600 nm with 600 μL quartz normal-melting-point agarose. Cells were lysed overnight at
cuvettes. Unless otherwise specified, all spectra were taken at 4 °C in cold alkaline lysis buffer (1% fresh Triton X-100, 2.5 M
37 °C in 10 mM sodium phosphate buffers.
NaCl, 100 mM EDTA, and 10 mM Tris-HCl, pH 10.0). The
For determination of the quantum efficiency of fluore- agarose gels were immersed into alkaline electrophoresis
scence (Yu), quinine sulfate in water (Ys = 0.54) was used as a buffer (300 mM NaOH and 1 mM EDTA at pH > 13) for 30 min,
standard. Values were calculated according to the following and then subjected to electrophoresis in the same buffer at
equation.
25 V and 300 mA for 40 min. The gels were then rinsed with
neutralization buffer (0.4 M Tris-HCl, pH 7.5) three times and
left to air-dry, after which they were stained with 10 μg mL−1
propidium iodide and incubated for 20 min. Finally, images
Fu As
ꢀ
Yu ¼ Ys ꢀ
Fs Au
where s means standard, u means sample, A means absor-
bance at the excitation wavelength, F means integrated area
under the fluorescence spectra on an energy scale.
were analyzed using
microscope.
a Nikon confocal laser scanning
γ-H2AX Immunofluorescence assay
Cell culture
Cells plated on a 35 mm glass-bottomed culture dish were
HeLa human cervical carcinoma cells (CCTCC, China) were fixed for 20 min and then washed in 0.5% Triton X-100/PBS.
cultured in MEM (Hyclone, China) supplemented with 10% The cells were then treated with permeabilization buffer
FBS, penicillin (100 units per mL), and streptomycin (100 μg [20 mmol L−1 Tris-HCl (pH 8), 0.5% Triton X-100, 50 mmol
mL−1). All the cells were maintained in a humidified atmos- L−1 NaCl, 3 mmol L−1 MgCl2, and 300 mmol L−1 sucrose] for
phere of 5/95 (v/v) of CO2–air at 37 °C.
15 min at 37 °C, washed twice with PBS, and then stained with
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Acknowledgements
The authors thank the National Basic Research Program of
China (973Program) (2012CB720600, 2012CB720603, 2012CB
720605), the National Science of Foundation of China (No.
91213302, 30973605, 21072115), the National Grand Program
on Key Infectious Disease (2012ZX10003002-014). Supported
by Program for Changjiang Scholars and Innovative Research
Team in University (IRT1030).
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