Molecular isomerization triggered by H2S to an NIR accessible first direct visualization of Ca2+-dependent production in living HeLa cells

Article abstract of DOI:10.1039/c9tb01885a

Few studies determined the role of intracellular labile Ca²⁺ in H₂S homeostasis. Undoubtedly, fluorescent probes are powerful tools for exploring the question because of their unique advantages: non-destruction, visualization, and mu

Full text of DOI:10.1039/c9tb01885a

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Molecular isomerization triggered by H2S to
an NIR accessible first direct visualization of
Cite this: J. Mater. Chem. B, 2019,
7, 6855
Ca2+-dependent production in living HeLa cells†
b
a
Ying Wen,a Fangjun Huo,
Junping Wanga and Caixia Yin
*
Few studies determined the role of intracellular labile Ca2+ in H2S homeostasis. Undoubtedly,
fluorescent probes are powerful tools for exploring the question because of their unique advantages:
non-destruction, visualization, and multi-levels imaging. Herein, a near-infrared (lem = 687 nm) and
methylene blue chromophore-based fluorescent probe (MB1) for H2S was rationally developed. Based
on its high sensitivity and selectivity, MB1 was employed to image the concentration change of H2S,
upon stimulating it with ionomycin (a specific calcium ionophore). We found that the intracellular labile
Ca2+ acted as a promotor for H2S production in living cells. Furthermore, cystathionine g-lyase (CSE)
might have functioned as a positive mediator of Ca2+-dependent H2S production. These direct and
visible links for H2S/Ca2+ will help us to understand the complex signaling in a better way.
Received 2nd September 2019,
Accepted 29th September 2019
DOI: 10.1039/c9tb01885a
rsc.li/materials-b
Thus, the development of fluorescent probes is of utmost impor-
tance. In this process, the selection of a fluorophore and the design
Introduction
Hydrogen sulfide (H2S), the third gasotransmitter,1,2 is produced of a reaction site need to cater to the probe’s practical applications.
enzymatically by cystathionine g-lyase (CSE), cystathionine Herein, via a smart design, the molecular isomerization and self-
b-synthase (CBS), or the cooperative effect of cysteine aminotrans- immolative triggered by H2S was employed in a near-infrared
ferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3-MST).3 region (lem = 687 nm) methylene blue dye with excellent biocom-
Alternate pathways for the formation of H2S is nonenzymatic path- patibility and optical performance,28,29 to construct an H2S probe
ways, such as changing from persulfide at the expense of reductants. (denominated in MB1) with enhanced sensitivity and selectivity.
H2S can regulate the insulin release,4 induce angiogenesis,5 and The presence of high concentrations of calcium ions did not
protect neurons from the oxidative stress,6 which has been reported interfere with the turn-on response of MB1 for H2S. Therefore,
to associate with diseases, such as liver cirrhosis,7 Alzheimer’s MB1 was used to detect the levels of H2S upon incubating the cells
disease,8 diabetes,9 Down syndrome,10 and hypertension.11
with ionomycin (a specific calcium ionophore). The cell imaging
Calcium ion (Ca2+), as a second messenger, was involved in a experiments clearly suggested that the levels of H2S increased
broad spectrum of intracellular signal transduction. Although H2S or sharply as the concentration of Ca2+ concentration increased.
Ca2+ signal transduction pathways have been characterized, few Namely, the intracellular labile Ca2+ acted as a promotor of H2S
studies examined the relationship between H2S and Ca2+ levels in production. Furthermore, with the assistance of MB1, it was found
living cells. As reported, the specific enzymes involved in the H2S that CSE might have played an important ‘mediator’ role. Thus,
production, such as CAT12,13 and CSE,14,15 were regulated by the this data will help us to understand the complex pathway interac-
intracellular calcium/calmodulin. However, a similar observation can tions between Ca2+ and H2S in living cells in a better way.
not be obtained by other laboratories.1 Thus, the role of intracellular
labile Ca2+ in the H2S hemostasis is considered ambiguous.
There is no denying that direct observation and image analysis
Experimental section
Materials
is one of the most persuasive evidences to solve this problem.16–27
a Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of
Education, Key Laboratory of Materials for Energy Conversion and Storage of
Shanxi Province, Institute of Molecular Science, Shanxi University,
All the starting materials were obtained from commercial
suppliers. S-Nitroso-N-acetyl-DL-penicillamine (SNP) was purchased
from Sigma-Aldrich (St Louis, MO, USA). 4-Amino benzene metha-
nol was purchased from Alfa Aesar (Ward Hill, MA, USA).
Ionomycin, 4-(6-acetoxymethoxy-2,7-dichloro-3-oxo-9-xanthenyl)-
40-methyl-2,20(ethylenedioxy)dianiline-N,N,N0,N0-tetraacetic acid
Taiyuan 030006, China. E-mail: yincx@sxu.edu.cn
b Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
† Electronic supplementary information (ESI) available: The details of character-
ization of compounds and additional data. See DOI: 10.1039/c9tb01885a
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tetrakis(acetoxymethyl) ester (Fluo-3AM), CCK-8, and cell culture 14 mm glass coverslips overnight, washed with PBS, and then
reagents were purchased from Beyotime Co., Ltd (Shanghai, incubated with MB1 (5 mM, 30 min) and/or Fluo-3 AM (1 mM,
China).
20 min) at 25 1C. After washing three times, the cells were
subjected to CLSM imaging. The red channel (690 ꢁ 30 nm)
was used to collect the MB1 signal under 633 nm as the excitation
Instruments
1H NMR (600 MHz) spectra and the high-resolution mass wavelength. In the detection experiments of exogenous and
spectra (HR-MS) were recorded on a Bruker Avance III HD endogenous H2S, the MB1-loaded HeLa cells were pre-incubated
spectrometer and AB Sciex Triple TOF 5600 instrument, respec- with SNP (100 mM, 30 min) or Na2S (160 mM, 30 min). In the Ca2+-
tively. The UV-visible spectra and steady-state emission experi- triggered experiments, the MB1-loaded or Fluo-3 AM-loaded HeLa
ments were recorded on a Hitachi U-3900 spectrometer and a cells were pre-incubated with 200 mM CaCl2 or 1 mM ionomycin for
Hitachi F-7000 spectrometer, respectively. The cell imaging 1.5 h. The green channel was set at 525 ꢁ 25 nm (lex = 488 nm) for
experiments were performed using a Zeiss LSM880 Airyscan Fluo-3AM.
laser confocal scanning microscope. The in vivo imaging assays
were performed on the Bruker In-Vivo FX Pro small animal
optical imaging system.
In vivo fluorescence images of BALB/c nude mice
BALB/c-nude mice were purchased from Sparford Biotechnology
Co., Ltd (Beijing, China). All the animal experiments were per-
formed by following the protocols approved by the Radiation
Protection Institute of Drug Safety Evaluation Center in China
(Production license: SYXK (Jin) 2018-0005). 50 mL of MB1 (1 mM)
and 50 mL of Na2S (1 mM) were injected into the right tibiotarsal
joints (right ankles) of 6–8 week-old BALB/c Nude Mice. The
imaging assays were performed in a small animal optical imaging
system, with an excitation filter of 630 nm and an emission filter
of 700 nm.
Synthesis details of MB1
The synthesis route of MB1 is shown in Scheme S1 in the ESI.†
C1. C1 was synthesized as per the previously reported method.30
FDOCl-2. FDOCl-2 was the gift of Professor Tao Yi and Dr Peng
Wei (Department of Chemistry, Fudan University, Shanghai,
China).25
MB1. C1 (70 mg, 0.47 mmol), FDOCl-2 (140.1 mg, 0.40 mmol),
Na2CO3 (124.4 mg, 1.17 mmol), and DMAP (50.7 mg, 0.4 mmol)
were dissolved in 20 mL anhydrous DCM. The reaction mixture
was cooled to 0 1C and stirred overnight under nitrogen. The
reaction was monitored by TLC. The product was purified via
column chromatography using ethyl acetate/petroleum ether
(1/4, v/v) as an eluent to obtain MB1 as a white powder. Yield
Results and discussion
Synthesis of MB1
1
40%; H NMR (600 MHz, CDCl3) d 7.33 (d, J = 7.7 Hz, 4H), 7.00
For constructing specifically the H2S-responsive probe, we
selected methylene blue as a fluorescent chromophore. A
previously reported reaction strategy of H2S and aryl azides to
amines was adopted.34,35 The 4-azidobenzyl group, as a self-
immolative aryl azide linker, was chosen to link to methylene
blue. First, we synthesized 4-azido benzene methanol from
4-amino benzene methanol and NaN3. Then, the condensation
reaction between 4-azido benzene methanol and the carbamoyl
chloride derivative of methylene blue (FDOCl-2) was employed
to obtain MB1. The details of the characterization of MB1 are
shown in Fig. S1–S3 of the ESI.†
(d, J = 7.8 Hz, 2H), 6.66 (s, 2H), 6.60 (d, J = 7.8 Hz, 2H), 5.18 (s, 3H),
2.93 (s, 12H). 13C NMR (151 MHz, DMSO) d 153.52 (s), 139.06 (s),
133.26 (s), 131.97 (s), 129.49 (s), 127.48 (s), 126.97 (s), 119.15 (s),
110.86 (s), 109.72 (s), 66.55 (s), 48.60 (s). HR-MS: for C24H24N6O2SNa+
[M + Na]+ calc. 483.1579, found 483.1573.
Fluorescence quantum yield determination
The fluorescence quantum yield (FF) was estimated by a
comparative method, where unsubstituted Zn(II) phthalo-
cyanine (ZnPc) was used as the ref. 31 The reference value of FF
was found to be 0.18 in DMSO for ZnPc.32 The fluorescence
quantum yield was calculated using: FF = FF(Std) ꢀ (AStd/A) ꢀ
(F/FStd) ꢀ (n2/nStd2), where the subscripts Std refer to the standard
Capability of MB1 to detect H2S in the solution
sample. F and FStd are the areas under the fluorescence emission To guide the follow-up cell experiments, we first examined the
curves of the sample and the standard, respectively. A and AStd are spectral properties of MB1 in Fig. 1. 5 mM of MB1 (DMSO : PBS,
the respective absorbances of the sample and standard at the 1 : 1, v/v) did not show any obvious absorption and fluorescent
excitation wavelengths. The n and nStd are the refractive indices of emission peak. However, after adding H2S (donors: 160 mM
the solvents used for the sample and standard, respectively.33 The Na2S), MB1 displayed one major absorption band in the wave-
absorbance of the solutions at the excitation wavelength ranged length range of 550 nm to 700 nm (centered at 666 nm,
between 0.1 to 0.02.
e = 20 600 Mꢂ1 cmꢂ1, Fig. 1A), which was observed to increase
with time, thereby suggesting that the chemical structure of
MB1 has changed. Moreover, the high-resolution mass spectra
analysis (Fig. S4, ESI†) confirmed that the new product was
Cell culture and confocal laser scanning microscope (CLSM)
imaging
The HeLa cells were cultured in RPMI 1640 media supplemented methylene blue, which was recovered via the self-immolative
with the 10% fetal bovine serum at 37 1C in a humidified release from MB1 and molecular isomerization after reduction
atmosphere containing 5% CO2. The cells were plated on by H2S (Scheme 1).
6856 | J. Mater. Chem. B, 2019, 7, 6855--6860
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intensities at 687 nm (F687, Fig. 1C). The detection limit was
calculated to be 0.32 mM by using the formula of 3s/k, where k is
the slope in Fig. 1C, and s is the standard deviation of the blank
samples.
Importantly, MB1 exhibited high chemoselectivity in the
spectroscopic response. When treated with various bio-
molecules, including metal ions (Ca2+, Mg2+, K+, and Na+),
amino acid (L-Cys, L-Ala, and L-Hcy), reactive sulfur species
(S2O32ꢂ, SO42ꢂ, and SCNꢂ), GSH, H2O2, and HClO, F687 of the
probe exhibited no changes, compared with a 49.2-fold increase
upon the stimulation of Na2S (Fig. 1D). Even in the presence of the
interfering species, particularly Ca2+, MB1 still showed an excellent
response to H2S. Thus, these results demonstrated that MB1 was a
highly sensitive and selective probe towards H2S.
Detection of H2S with MB1 in living cells
Fig. 1 The changes in the absorption (A) and fluorescence (B) spectra of
MB1 (5 mM, DMSO : PBS, 1 : 1, v/v) over time after addition of Na2S (160 mM).
Insets of (A): the color changes of MB1 in the absence and presence of
H2S. (C) Plot of fluorescence intensity at 687 nm (F687) for MB1 (5 mM) vs.
[Na2S] from 1 to 35 mM. Each data was collected after 1 h upon the addition
of Na2S. The error bars and average values are shown for experiments
repeated 3 times independently. (D) F687 of MB1 (5 mM) in response to
various other biologically relevant species after 1 h at 25 1C. Black bar of
Prior to the imaging application in living cells, the CCK-8 assay
was performed to verify the low acute toxicity of MB1 to living
HeLa cells. The viabilities were found to be 496% at 5 h or 10 h
when 1–10 mM of MB1 was incubated (Fig. S7, ESI†), thereby
clearly suggesting that MB1 possessed excellent biocompatibility.
We next assessed whether the probe had sufficient sensitivity
to visualize the exogenous and endogenous H2S in HeLa cells
using a confocal laser scanning microscope (CLSM). The MB1-
loaded (5 mM, 25 1C, 30 min) HeLa cells were imaged from the red
fluorescence channel (690 ꢁ 30 nm). Upon pre-incubation with
160 mM of Na2S for 30 min, the HeLa cells (Fig. 2a7) showed
strong red fluorescence, clearly indicating the good cell-
membrane permeability and H2S response ability of MB1 in living
cells. Then, to verify the high sensitivity of the probe, MB1 was
employed to monitor the H2S generated endogenously in living
cells (Fig. 2a4). Moreover, SNP (a commercial NO donor) was used
to stimulate the generation of endogenous H2S.36 The increased
fluorescence signal was observed in MB1-treated HeLa cells pre-
incubated with 100 mM of SNP for 30 min. These results indicated
that MB1 was capable of visualizing endogenous H2S generation.
MB1 can also be used to correlate different levels of H2S in vivo
with the intensity being in the NIR emissions range (Fig. S8, ESI†).
After verifying the high selectivity and sensitivity, MB1 was
1 was blank; 2 to 15 represent 500 mM SO42ꢂ, S2O32ꢂ, SCNꢂ, Ca2+, Mg2+
,
K+, Na+, GSH, L-Me, L-Cys, L-Ala, L-Hcy, H2O2, and HClO, respectively. Red
columns represent the extra addition of 160 mM Na2S. lex = 600 nm.
In addition, MB1 showed a fluorescence turn-on response
toward H2S. Upon treatment of 5 mM of MB1 with H2S, a near-
infrared fluorescence emission ranging from 632 nm to 775 nm
(majored in 687 nm, Fig. 1B) was observed, which drastically
increased over time, with 600 nm excitation. After reacting with
H2S, the fluorescence quantum yield (FF) of MB1 increased
from less than 0.02 to 0.13. The same fluorescence spectral
experiments were performed without H2S (Fig. S5, ESI†), and it
was observed that the absorption and fluorescence spectra did
not show any change, which in turn indicated the stability of
MB1. The dynamic curve of MB1 is shown in Fig. S6 (ESI†).
The concentration of Na2S varying from 1 to 35 mM showed a
better linear relationship (R2 = 0.9931) with the fluorescence
used to directly evaluate the change in the H2S level of the Ca2+
-
treated cells. Compared with the only probe group (MB1, 5 mM,
30 min, Fig. 3b1), the red fluorescence signal (690 ꢁ 30 nm) in
the HeLa cells of Ca2+-pretreated (200 mM, 1.5 h) group
(Fig. 3c1) markedly increased. In addition, the corresponding
average fluorescence intensities of the cells shown in Fig. 3b1
and c1 presented statistical changes of p o 0.05. However,
upon co-incubating the cells with 200 mM of EDTA (metal ion
chelating agent), the red fluorescence intensity of the cells
stimulated by Ca2+ (Fig. 3e1, p o 0.05) was reduced signifi-
cantly. Further, taking into consideration that Ca2+ homeo-
stasis is tightly controlled in living cells, a universal Ca2+
fluorescent probe (Fluo-3 AM) was employed to demonstrate
that incubation with 200 mM CaCl2 can induce a slight increase
in the intracellular labile Ca2+ concentrations (Fig. 3c3). The
fluorescence signal of Fluo-3 AM was collected within the green
channel (525 ꢁ 25 nm), under 488 nm as an excitation
Scheme 1 The chemical structure, response mechanism, and potential
imaging applications of MB1.
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wavelength. Therefore, these data indicated that in HeLa cells,
the stimulation of Ca2+ could induce the H2S explosion.
Ionomycin was employed to further verify the above result,
which is known to raise the intracellular level of Ca2+ via
allowing direct Ca2+ influx across the cellular membrane.37,38
As expected, the pre-incubation with 1 mM of ionomycin for
1.5 h induced strong green fluorescence in Fluo-3 AM-loaded
cells, as shown in Fig. 3d3. Compared with that of the green
fluorescence in Fig. 3b3, the average fluorescence intensities of
cells in Fig. 3d3 increased by 10.03 (p o 0.01). Namely, the
intracellular Ca2+ concentration increased sharply. Moreover,
after stimulation with ionomycin, a significant increase was
obvious in the red fluorescence intensity of MB1-loaded HeLa
cells. The average fluorescence intensities of cells in Fig. 3d1
was about 17.64 times ( p o 0.001) than that in Fig. 3b1. As
shown in Fig. S9 (ESI†), the ionomycin itself neither induced
the increase in the fluorescence intensity of the MB1 probe nor
interfered with the response to H2S. Therefore, these results
indicated that the H2S production was related to Ca2+ stimula-
tion in the HeLa cells.
Fig. 2 (left) The CLSM images of HeLa cells loaded with 5 mM of MB1. The
cells were pre-incubated with SNP (100 mM, 30 min) or Na2S (160 mM,
30 min); red channel, 690 ꢁ 30 nm; lex = 633 nm; scale bar = 10 mm. (right)
Corresponding average fluorescence intensities of the respective cells in
the left images. Statistical analyses were employed with the Student’s t-test
(n = 3). Compared with the control group: *p o 0.05, **p o 0.01, and error
bars are ꢁS.D.
Next, we further explored the correlation between the Ca2+
and H2S levels. HeLa cells co-loaded with 5 mM of MB1 and
1 mM of Fluo-3 AM were additionally pre-incubated with 1 mM of
Fig. 3 The CLSM images of (A) MB1 (5 mM, 30 min) or (B) Fluo-3 AM (1 mM,
20 min)-loaded HeLa cells. (a1–a4) control group; before being loaded
with MB1 or Fluo-3 AM, the HeLa cells were pre-incubated with (b1–b4)
only PBS; (c1–c4) CaCl2; (d1–d4) ionomycin; (e1–e4) CaCl2 and EDTA at
37 1C for 90 min. CaCl2: 200 mM; ionomycin: 1 mM; EDTA: 200 mM; Red
channel, 690 ꢁ 30 nm, lex = 633 nm for MB1; Green channel, 525 ꢁ
25 nm, lex = 488 nm for Fluo-3 AM; Scale bar = 10 mm. (C) or (D)
Corresponding average fluorescence intensities of cells in (A) or (B),
respectively. Statistical analyses were employed with Student’s t-test (n =
3). Compared with the control (a1 or a3) group: *p o 0.05, **p o 0.01, and
***p o 0.001. Compared with the group (b1 or b3) having only probe:
#p o 0.05, ##p o 0.01, and ###p o 0.001. Compared with the CaCl2-
treated (c1 or c3) group; &p o 0.05, &&p o 0.01, and error bars are ꢁS.D.
Fig. 4 The CLSM images of MB1 (5 mM) and Fluo-3 AM (1 mM)-loaded
HeLa cells. The cells were pre-incubated with ionomycin (1 mM) for 0 min,
10 min, 25 min, 45 min, and 60 min. Red channel, 690 ꢁ 30 nm; lex = 633 nm
for MB1; Green channel, 525 ꢁ 25 nm; lex = 488 nm for Fluo-3 AM;
Scale bar = 10 mm. (B) and (C) Corresponding average fluorescence intensities
of cells. Statistical analyses were employed with Student’s t-test (n = 3).
Compared with the 0 min group: *p o 0.05, **p o 0.01, ***p o 0.001,
and ****p o 0.0001. Compared with the ionomycin (ꢂ) group: #p o 0.05,
##p o 0.01, ###p o 0.001, ####p o 0.0001, and error bars are ꢁS.D.
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ionomycin for different time (0 min, 10 min, 25 min, 45 min, Conclusion
and 60 min) (Fig. 4A). The control group (without ionomycin) is
In summary, we synthesized a highly selective and sensitive
fluorescence probe (MB1) to visualize the change of H2S levels
in living cells, and to determine the role of intracellular Ca2+ in
the H2S homeostasis. Using MB1, we imaged the excess H2S
levels in the Ca2+-triggered HeLa cells and studied the depen-
dent effect between them. Furthermore, we provided a potential
pathway of the H2S explosion mediated by Ca2+ in living cells,
which provides new insights into the complex signal transduc-
tion in living cells.
shown in Fig. S10 (ESI†). We found that the fluorescence
intensities of the red channel (for MB1, Fig. 4a1 to e1) and
green channel (for Fluo-3 AM, Fig. 4a2 to e2) increased with an
increase in the ionomycin incubation time. Compared with the
0 min group, the average fluorescence intensities of the cells
incubated with ionomycin for 10 min in red (Fig. 4b1) and
green (Fig. 4b2) channels increased 5.86 ( p o 0.05) and 5.12
( p o 0.01) times, respectively. At 10 min, there were statistically
significant differences between ionomycin (+) group and iono-
mycin (ꢂ) group (Red channel intensity: Fig. 4B, p o 0.05;
Green channel intensity: Fig. 4C, p o 0.01). Subsequently, with
the increase in the ionomycin incubation time, the green
channel fluorescence corresponding to Fluo-3 AM increased
considerably. Moreover, the red fluorescence presented a slow
upward trend. At 60 min, compared with the 0 min group, the
average fluorescence intensities of the cells in red (Fig. 4e1) and
green (Fig. 4e2) channels increased by 24.40 (Fig. 4C, p o 0.05)
and 50.96 (Fig. 4C, p o 0.001) times, respectively. These results
indicate that H2S production was dependent on the Ca2+ level
in the HeLa cells. Additionally, similar results of Ca2+ and H2S
levels were observed by direct incubation of the cells with
200 mM of CaCl2 (Fig. S11, ESI†).
Next, MB1 was also used to uncover the pathways that result
in H2S production upon Ca2+ stimulation of the HeLa cells.
For this purpose, the HeLa cells were first pre-incubated
with aminooxyacetic acid (AOAA, 100 mM, 3 h), a potent
inhibitor of CBS.39 Then, MB1 was used to image the change
in the cellular H2S level upon Ca2+ (200 mM, 1 h) stimulation.
AOAA did not inhibit the increase in the average cellular
fluorescence intensity of MB1 (Fig. 5A). However, the CSE
inhibitor, DL-propargylglycine (PAG),19 absolutely inhibited
the turn-on response of MB1 towards H2S. Moreover, the
average cellular fluorescence intensity decreased from 3.8
to 1.3 ( p o 0.01, Fig. 5B). This indicated that CSE might
contribute to the observed H2S generation upon Ca2+
stimulation.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank the National Natural Science Foundation of China
(No. 21705102, 21775096), the Shanxi Province Science Founda-
tion for Youths (No. 201701D221061) Shanxi Province Founda-
tion for Returness (No. 2017-026), One hundred people plan of
Shanxi Province, Shanxi Province ‘‘1331 Project’’ Key Innova-
tion Team Construction Plan Cultivation Team (2018-CT-1),
Shanxi Province Foundation for Returness (2017-026), Shanxi
Collaborative Innovation Center of High Value-added Utiliza-
tion of Coal-related Wastes, 2018 Xiangyuan County Solid
Waste Comprehensive Utilization Science and Technology Pro-
ject 2018XYSDJS-05, China Institute for Radiation Production
and Scientific Instrument Center of Shanxi University (201512).
Ying Wen also thanks Dr Peng Wei and Professor Tao Yifrom
Fudan University for the gift of FDOCl-2.
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Fig. 5 (A) The CLSM images of the Ca2+-dependent H2S production
pathways. Before imaging, MB1 (5 mM, 30 min)-loaded HeLa cells were
pre-incubated with AOAA or PAG (100 mM, 3 h), and then stimulated with
CaCl2 (200 mM, 1 h). Red channel, 690 ꢁ 30 nm, lex = 633 nm for MB1;
Scale bar = 10 mm. (B) Corresponding average fluorescence intensities of
cells in (A). Statistical analyses were employed with Student’s t-test (n = 3).
Compared with the a1 group: **p o 0.01. Compared with the Ca2+
-
treated (b1) group: ##p o 0.01 and error bars are ꢁS.D.
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