Full Papers
DOI: 10.1002/cbic.201600060
Fast-Response Turn-on Fluorescent Probes Based on
Thiolysis of NBD Amine for H2S Bioimaging
Runyu Wang+,[b] Zhifei Li+,[a] Changyu Zhang,[a] Yanyan Li,[b] Guoce Xu,[c] Qiang-Zhe Zhang,[c]
Lu-Yuan Li,[c] Long Yi,*[a] and Zhen Xi*[b]
Hydrogen sulfide (H2S) is an important endogenous signaling
molecule with multiple biological functions. New selective fluorescent turn-on probes based on fast thiolyling of NBD (7nitro-1,2,3-benzoxadiazole) amine were explored for sensing
H2S in aqueous buffer and in living cells. The syntheses of both
probes are simple and quite straightforward. The probes are
highly sensitive and selective toward H2S over other biologically relevant species. The fluorescein-NBD-based probe showed
65-fold green fluorescent increase upon H2S activation. The
rhodamine-NBD-based probe reacted rapidly with H2S (t1/2
… 1 min) to give a 4.5-fold increase in red fluorescence. Moreover, both probes were successfully used for monitoring H2S in
living cells and in mice. Based on such probe-based tools, we
could observe H2O2-induced H2S biogenesis in a concentrationdependent and time-dependent fashion in living cells.
Introduction
H2S-mediated reduction,[6–8] nucleophilic addition/substitution,[9] and dual-nucleophilic addition/substitution[10] have been
employed for the development of fluorescent probes. In spite
of the success of these fluorescent probes for H2S biology,
most of reaction-based probes show delayed responses (typically > 10 min) toward H2S.[6–10] Recently, we explored fluorescent H2S probes based on selective fast thiolysis of 7-nitro1,2,3-benzoxadiazole (NBD) amine.[9a, f, 11] These probes were
based on coumarin derivatives, which have relatively short fluorescent wavelengths. In this work, we used selective thiolysis
of NBD amine to develop new fast-response H2S probes with
fluorescein and rhodamine as the fluorophores. We successfully obtained two fluorescent probes for fast and selective detection of H2S, both in aqueous buffer and in vivo.
Hydrogen sulfide (H2S) is an important endogenous signaling
molecule with multiple biological functions.[1–3] H2S is produced
by three distinct enzymes in different organs and tissues: cystathionine b-synthase (CBS), cystathionine g-lyase (CSE), and 3mercaptopyruvate sulfurtransferase (3-MPST)/cysteine aminotransferase (CAT).[2] Studies have shown that H2S levels are
associated with numerous diseases, including Alzheimer’s disease, Down syndrome, diabetes, and liver cirrhosis.[4] Despite
recognition of the link between H2S and numerous physiological and pathological processes, many of its underlying molecular events remain largely unknown. Therefore, there is significant research value in developing efficient methods for detecting H2S in living biological systems.
Compared with other methods,[5] fluorescent probes should
be excellent for in-situ and real-time monitoring of H2S in biological samples, because of their non-destructive sensing of
bio-targets and easy detection.[6–12] Organic reactions involving
Results and Discussion
The thiolysis of NBD amine is a useful reaction for the design
of fluorescent H2S probes.[9a] The NBD moiety can quench the
fluorescence of the fluorophore in aqueous solution, and it is
released after the thiolysis of the NBD moiety by H2S. Based on
this strategy, new fluorescent probes 1 and 2 were simply
prepared by coupling fluorescein or rhodamine B to an NBDbased moiety 3 (Scheme 1). Both probes were well characterized by 1H NMR, 13C NMR, and HRMS (see the Supporting Information).
We then examined the spectral properties of the probes in
PBS buffer. The absorbance of 1 displayed a maximum at
502 nm (Figure 1 A). After treatment with H2S (using Na2S as an
equivalent), the maximum peak decreased slightly, and a new
one appeared at 530 nm upon treatment with H2S (Figure 1 A).[13] The fluorescent tests were further carried outin PBS
buffer (Figure 1 B). Probe 1 showed weak fluorescence (quantum yield f= 0.01), thus indicating that fluorescein fluores-
[a] Z. Li,+ C. Zhang, L. Yi
State Key Laboratory of Organic-Inorganic Composites
Beijing University of Chemical Technology (BUCT)
15 Beisanhuan East Road, Chaoyang District, Beijing 100029 (China)
E-mail: [email protected]
[b] R. Wang,+ Y. Li, Prof. Dr. Z. Xi
Department of Chemical Biology
State Key Laboratory of Elemento-Organic Chemistry
National Engineering Research Center of Pesticide (Tianjin)
Collaborative Innovation Center of Chemical Science and Engineering
(Tianjin)
Weijin Road 94, Nankai District, Tianjin 300071 (China)
E-mail: [email protected]
[c] G. Xu, Q.-Z. Zhang, Prof. Dr. L.-Y. Li
State Key Laboratory of Medicinal Chemical Biology, Nankai University
Weijin Road 94, Tianjin 300071 (China)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201600060.
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Scheme 1. Synthesis of probes 1 and 2 and their reactions with H2S.
BSA, and yeast lysate. The fluorescence enhancement for each
tested molecule was negligible relative to that for H2S (Figure 2 C), thus implying that 1 can selectively detect H2S in
biological samples. Additionally, we tested the fluorescence
response of 1 to H2S at different pH values (Figure 2 D); the
probe is functional over pH 6.0 to 8.5.
We also tested probe 2. Its absorbance displayed a maximum
at 567 nm with a side peak at 475 nm (Figure 3 A). After reacting with H2S, the maximum peak increased and the side peak
decreased, while a new peak appeared at 530 nm.[13] HRMS in-
Figure 1. Time-dependent spectra of fluorescein-NBD-based probe 1 in the
presence of H2S. A) Absorption spectra of 10 mm 1 with 1 mm H2S. B) Fluorescent spectra of 1 mm 1 with 100 mm H2S. All tests were performed at 25 8C
in PBS (pH 7.4).
cence was significantly quenched by FRET (Figure S2 in the
Supporting Information). A 65-fold fluorescent increase was
observed for 1 in the presence of H2S (f= 0.64). The reaction
kinetics of a probe with H2S is important for its biological applicability on account of the rapid catabolism of H2S under
physiological conditions. To obtain the reaction kinetics, timedependent fluorescence signals were acquired for data analysis
(Figure S1). For 1, the pseudo-first-order rate, kobs, was 2.8 Õ
10¢3 s¢1 by fitting the data to a single exponential function.
The reaction rate k2 (k2 = kobs/[H2S]) was calculated as 28 m¢1 s¢1,
thus indicating that 1 is a fast-response H2S probe.
Encouraged by these results, we studied the fluorescence response of 1 with different concentrations of H2S (0–100 mm;
Figure 2 A). The fluorescence intensity was linearly related to
H2S concentration over 0–30 mm (Figure 2 B). Notably, the detection limit of 1 was determined to be 0.057 mm, based on
the 3s/slope method.[14] A major challenge for H2S detection in
biological systems is to develop a selective probe that exhibits
specificity for H2S over other cellular molecules, especially millimolar biothiols. To gain selectivity information, probe 1 was
incubated with various biologically relevant species, including
reactive sulfur species (biothiols, SO32¢, SO42¢, S2O32¢), reactive
oxygen species (H2O2 + NaClO), NaNO2, cations (Zn2 + , Fe3 + ),
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Figure 2. Fluorescent spectra of 1 with H2S. A) Fluorescent emission of
1 (1 mm) with different concentrations of H2S in PBS (pH 7.4) at 25 8C for
20 min. B) Linear relationship between fluorescence intensity at 530 nm and
H2S concentration (0–30 mm). C) Fluorescence spectra of 1 (1 mm) incubated
with various compounds (100 mm H2S, SO42¢, SO32¢, S2O32¢, NO2¢, H2O2,ClO¢ ,
Zn2 + and Fe3 + ; 1 mm Hcy; 5 mm GSH; 10 mg mL¢1BSA; 1 mg mL¢1yeast
lysate, in PBS with 10 % SDS for 20 min. D) Emission at 530 nm of 1 (1 mm) at
the indicated pH in the absence or presence of H2S (100 mm). Each reaction
was performed in PBS for 20 min.
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Figure 3. Time-dependent spectra of rhodamine-NBD-based probe 2 in the
presence of H2S. A) Absorption spectra of 10 mm 2 with 1 mm H2S. B) Fluorescent spectra of 1 mm 2 with 100 mm H2S. All tests were performed at 25 8C
in PBS (pH 7.4).
dicated cleavage of 2 to produce 4 (Supporting Information).
Probe 2 showed weak fluorescence at 589 nm (f= 0.18; Figure 3 B). After H2S treatment, the intensity increase at 589 nm
(4.5-fold; f 0.36) was much smaller than that for 1. Quenching
of rhodamine B fluorescence by the NBD group might depend
on the photo-induced electron transfer (PET) effect from the
nitro group in the NBD moiety; this should be significantly
influenced by the distance between the fluorophore and the
quencher. However, the reaction rate (k2) for 2 was calculated
as 113 m¢1 s¢1 (Figure S1), which makes it is among the fastest
of the H2S probes,[6] and comparable with the probe with aldehyde-helped thiolysis reported by Feng and co-workers.[9 h]
Probe 2 has a positive charge at a nitrogen atom in rhodamine, and this might help to accumulate HS¢ anions (the
major component of H2S at pH 7.4) around the probe to
enhance the thiolysis rate. A similar observation was noted by
Wu and Tang for fluoride fluorescent probes.[15]
In order to further understand the quenching effect and reaction rate of 2, we prepared a control molecule (6) with a
longer linker between rhodamine and NBD (Figure S3). The
fluorescence increase of 6 upon H2S addition was only 1.2-fold
(possibly because of a weaker PET effect) and it showed slower
thiolysis (69 m¢1 s¢1) than for 2, but this was still 2.4-fold faster
than for 1, because electrostatic interactions decrease with distance.
Encouraged by these results, we studied the fluorescence response of 2 with different concentrations of H2S (0–100 mm;
Figure 4 A). The fluorescence intensity was linearly related to
concentration of over 0 to 30 mm (Figure 4 B), and the detection limit was 0.58 mm, based on the 3s/slope method. To
assess selectivity, it was incubated with various species as described for 1. Only SO32¢ showed limited fluorescence response
(Figure 4 C),[9a] thus indicating that 2 can selectively detect H2S
and with little cross-reaction with other biologically relevant
species. Probe 2 was functional over pH 6.0–8.5 (Figure 4 D).
The cytotoxicity of both probes was evaluated on HEK293
cells by an MTT assay (Figures S4 and S5). Neither probe
showed cytotoxicity at 2 mm, thus implying that the probes are
suitable for H2S detection in living cells at this concentration.
To test the biological applicability of the probes, we examined whether they can be used to detect exogenous H2S in
living cells. HEK293A cells were treated with 1, washed with
PBS to remove excess probe, then incubated with H2S and
ChemBioChem 2016, 17, 962 – 968
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Figure 4. Fluorescent spectra of 2 with H2S. A) Fluorescent response of 2
with different concentrations of H2S in PBS (pH 7.4) at 25 8C for 6 min.
B) Linear relationship (r = 0.991) between fluorescence intensity at 589 nm
and H2S concentration. C) Fluorescence spectra of 2 (1 mm) incubated with
various analytes (100 mm H2S, SO42¢, SO32¢, S2O32¢, NO2¢, H2O2,ClO¢ , Zn2 + ,
and Fe3 + ; 1 mm Cys and Hcy; 5 mm GSH) for 6 min. D) Emission at 589 nm
of probe 2 (1 mm) at the indicated pH values in the absence or presence of
H2S (100 mm). Each reaction was performed in PBS for 6 min.
imaged by confocal fluorescence microscopy (Figure 5): green
fluorescence was observed; cells treated with only 1 did not
show fluorescence. We also observed exogenous H2S with
probe 2 (Figure S6).
Next, a fluorescent co-localization assay with MitoTracker
Green FM and 2 was performed (Figure 6). Probe 2 co-localized
with MitoTracker Green FM (Pearson coefficient 0.929), thereby
implying preferential distribution of 2 in mitochondria (Figure 6 C). Bright-field images show that the cells retained morphology after incubation with both probes (Figures 5 and 6),
thus also suggesting good biocompatibility for both probes.
These preliminary studies suggested that both probes could
be used for visualization of H2S in cells efficiently and selectively.
To test whether 1 and 2 can detect endogenous production
of H2S, cells were co-incubated with d-Cys or l-Cys and 1 or 2.
Our previous work indicated that both d-Cys and l-Cys can
induce enzymatic H2S production in the mitochondria of
HEK293 cells, and that this production can be inhibited by the
cysteine mimic propargylglycine (PPG).[8d] HEK293A cells displayed weak fluorescence when treated with 1 alone (Figure 7 A). After using chiral Cys to induce endogenous H2S production, significantly fluorescent enhancement was observed
(Figure 7 B, C); this was much brighter than for cells in the
presence of inhibitor PPG (Figure 7 D, E). The average fluorescence values of the images (Figure 7 F) imply that both d-Cys
and l-Cys induce H2S production in living cells and that H2S
biogenesis from d-Cys is more efficient than that from l-Cys.
Similar conclusions were obtained for 2 (Figures S8, S9). Taken
together, the results indicate that both probes can be used for
bioimaging endogenous H2S in living cells.
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Figure 7. HEK293 cells incubated with 1 and Cys. A) Cells incubated with
2 mm 1. B) Cells co-stimulated with 2 mm probe and 100 mm L-Cys for 60 min.
C) Cells co-stimulated with 2 mm probe and 100 mm d-Cys for 60 min.
D) Cells incubated with DL-PPG (50 mg L¢1) for 20 min, then co-stimulated
with 2 mm probe and 100 mm l-Cys for 60 min. E) Cells incubated with DLPPG (50 mg L¢1) for 20 min, then co-stimulated with 2 mm probe and 100 mm
d-Cys for 60 min. Scale bar: 50 mm. F) The average fluorescence intensity of
each image (lanes 1–5 for A–E, respectively).
Figure 5. Confocal microscopy images of exogenous H2S in living HEK293A
cells with 1. Cells were incubated with A) 1 (2 mm) for 30 min; B) 1 (2 mm) for
30 min and then Na2S (150 mm) for 60 min. Overlap images of fluorescence
and bright-field are shown below. Scale bar: 50 mm.
enous H2S production from living cells in a concentrationdependent fashion. In order to observe H2O2-induced H2S biogenesis in real-time, we used the fast-response probe 2 (Figures 8 C, S11, and S12). More H2S was produced with longer incubation time. Time-dependent H2S production by exogenous
H2O2 stimulation should help to further understand intracellular redox homeostasis.
Finally, we examined the suitability of the longer wavelength
probe for visualizing H2S in vivo. Mice were intraperitoneally injected with probe 2 and Na2S for 10 min (control mice, only
probe). The mice were imaged with a small animal in vivo
imaging system with a 535 nm excitation filter and a DsRed
emission filter. Figure 9 shows fluorescence images of living
mice with or without Na2S. Control mice showed a faint background fluorescence, whereas mice with Na2S injection
showed dramatically higher fluorescence (according to the
pseudocolor), thus indicating that 2 can serve as a fluorescent
agent for H2S imaging in vivo.
Figure 6. Confocal microscopy images of exogenous H2S in mitochondria
with 2. Living cells were incubated with Na2S (100 mm) for 30 min and then
co-stained by Mito-Tracker Green FM (0.25 mm) and probe 2 (2 mm) for
30 min. A) Image from bandpass 555–655 nm upon excitation at 546 nm;
B) Image from bandpass 500–530 nm upon excitation at 488 nm; C) Merged
image; D) Bright-field image. Scale bars: 50 mm.
Conclusion
Two new NBD-based fluorescent turn-on probes were developed for H2S detection in aqueous buffer and in living cells.
The probes are based on the fast and selective thiolyling of
the NBD amine bond; this could be a general design strategy
for H2S probes. Probe 1 showed 65-fold green fluorescent
increase upon H2S activation. Probe 2 could react rapidly with
H2S (t1/2 … 1 min) to give a 4.5-fold red fluorescence increase.
The higher reaction rate of 2 toward H2S might be attributable
to the positively charged nitrogen in rhodamine, which also
contributes to mitochondrial targeting. Both probes exhibited
excellent selectivity toward H2S over millimolar biothiols in
physiological buffer. Moreover, our probes were successfully
applied for bioimaging of H2S in living cells and in mice. The
To further demonstrate the biological applicability of the
NBD-based probes, we examined whether they can detect
H2O2-induced H2S in living cells. In our previous work, the first
H2O2–H2S dual-response probe was prepared for visualization
of H2O2-induced H2S in living cells for the first time.[16] Here,
when cells were treated with different concentrations of H2O2
followed by 1, fluorescent signals were observed (Figure 8, Figure S10). and employed for quantification of H2O2-induced H2S
production. The results indicated that H2O2 can induce endogChemBioChem 2016, 17, 962 – 968
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Figure 9. Representative fluorescence imaging of H2S in living mice. The
mice were injected with A) probe 2 (150 mm, 200 mL) for 10 min or with
B) probe 2 (150 mm, 200 mL) and then with Na2S (100 mm, 200 mL) for 10 min,
then imaged with an IVIS Lumina II (535 nm excitation filter, DsRed emission
filter).
ppm relative to the internal standard tetramethylsilane (Si(CH3)4 =
0.00 ppm) or residual solvent peaks (CDCl3 = 7.26 ppm; DMSO =
2.5 ppm). High-resolution mass spectra were obtained on a 6540
UHD Accurate-Mass Q-TOFLC/MS (Agilent Technologies). All spectroscopic measurements were performed in PBS (50 mm, pH 7.4)
containing DMSO (10 %). Stock solutions of compounds (10.0 or
1.0 mm) were dissolved in DMSO. UV-visible spectra were recorded
on a UV-3600 UV-VIS-NIR spectrophotometer (SHIMADZU, Japan).
Fluorescence study was carried out with an F-280 spectrophotometer (Tianjin Gangdong Sci & Tech., Tianjin, China). All measurements
were performed in a 3 mL cuvette with 2 mL solution.
Fluorescein-NBD-based probe (1): Fluorescein (86 mg, 0.26 mmol)
was dissolved in DMF (5 mL), and then HATU (296 mg, 0.78 mmol)
and DIPEA (268 mL, 1.56 mmol) were added. After 5 min of stirring,
3 (75 mg, 0.3 mmol) was added. The mixture was stirred at room
temperature overnight, then DMF was removed under reduced
pressure. The residue was purified by silica gel column chromatography to give 1 as a red solid (81 mg, 55 %). TLC (CH2Cl2/MeOH
100:8) Rf = 0.36; 1H NMR (600 MHz, [D6]DMSO): d = 8.48 (d, J =
9.1 Hz, 1 H), 7.73–7.69 (m, 3 H), 7.50 (dd, J = 6.8, 2.3 Hz, 1 H), 6.93 (d,
J = 9.2 Hz, 2 H), 6.54 (t, J = 7.8 Hz, 3 H), 6.49 (s, 2 H), 4.00 (br s, 4 H),
3.67 (br s, 2 H), 3.57 ppm (br s, 2 H); 13C NMR (151 MHz, [D6]DMSO):
d = 166.9, 156.1, 148.4, 145.2, 144.8, 144.7, 136.3, 135.1, 131.5,
130.6, 130.5, 129.7, 129.3, 127.4, 121.5, 114.1, 103.4, 103.2, 49.0,
48.3, 45.8, 40.8 ppm; HRMS (ESI): m/z calcd for C38H40N7O5 + :
564.1514 [M+
+H] + , found: 564.1518.
Figure 8. A) H2O2 induces H2S production in living cells as revealed by
probes 1 or 2. B) HEK293 cells were incubated with 1 for 30 min and then
with H2O2 (0–250 mm) for 30 min. Data are average fluorescence intensities
of each image. C) HEK293 cells were incubated with 200 mm H2O2 for 30 min
and then with 2 mm 2. Images were taken at the indicated time. Data are
average fluorescence intensity of each image.
bioimaging results indicated that our probes could monitor
Cys- and H2O2-induced endogenous H2S in living cells, thus implying that the NBD-based probes could be useful tools for
H2S biology. This work further highlights that thiolysis of the
NBD amine is a useful reaction for development of fluorescent
H2S probes.
Rhodamine-NBD-based probe (2): Rhodamine B (172 mg,
0.36 mmol) was dissolved in DMF (5 mL), then HATU (163 mg,
0.43 mmol) and DIPEA (148 mL, 0.86 mmol) were added. After
5 min of stirring, 3 (75 mg, 0.3 mmol) was added. The mixture was
stirred at room temperature overnight, then DMF was removed
under reduced pressure. The residue was purified by silica gel
column chromatography to give 2 as a dark red solid (199 mg,
94 %). TLC (CH2Cl2/MeOH 100:6) Rf = 0.4; 1H NMR (400 MHz,
[D6]DMSO): d = 8.51 (d, J = 9.1 Hz, 1 H), 7.81–7.76 (m, 3 H), 7.58–7.52
(m, 1 H), 7.21 (s, 1 H), 7.18 (s, 1 H), 7.11 (d, J = 2.1 Hz, 1 H), 7.09 (d,
J = 2.1 Hz, 1 H), 6.93 (d, J = 2.1 Hz, 2 H), 6.56 (d, J = 9.2 Hz, 1 H), 4.03
(br s, 4 H), 3.78 (br s, 2 H), 3.63 (q, J = 6.9 Hz, 8 H), 3.54 (br s, 2 H),
1.18 ppm (t, J = 6.9 Hz, 12 H); 13C NMR (100 MHz, [D6]DMSO): d =
Experimental Section
General: All chemicals and solvents used for synthesis were purchased from commercial suppliers and applied without further
purification. The progress of the reaction was monitored by TLC on
60F-254 pre-coated silica plates (250 mm; Merck Millipore), and
spots were visualized by basic KMnO4, UV light, or iodine. Silica gel
60 (100–200 mesh; Merck Millipore) was used for general column
chromatography. 1H NMR and 13C NMR spectra were recorded on
Bruker 400 and 600 spectrometers. Chemical shifts are reported as
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166.8, 157.1, 155.6, 155.1, 145.2, 144.7, 144.6, 136.2, 135.0, 131.7,
130.8, 130.5, 130.0, 129.9, 127.6, 121.6, 114.3, 113.1, 103.4, 95.9,
54.9, 49.3, 47.8, 45.6, 45.4, 41.0, 12.4 ppm; HRMS (ESI): m/z calcd
for C38H40N7O5 + : 674.3085 [M] + , found: 674.3065.
guidelines for animal experiment. The accreditation number of the
laboratory is SYXK(Jin) 2014-00003 promulgated by Tianjin Science
and Technology Commission. The intraperitoneal cavity of female
nude mice (6 weeks) were injected with 2 (150 mm, 200 mL) for
10 min. The mice were imaged on an IVIS Lumina II system (Xenogen/PerkinElmer), a small animal in vivo imaging system with
a 535 nm excitation filter and a DsRed emission filter. Next, Na2S
(100 mm, 200 mL) was injected into the intraperitoneal cavity for
10 min, and then images were collected.
Procedure of fluorescence measurement: PBS was degassed by
bubbling with N2 for 30 min. Stock solutions of Na2S (1–1000 mm)
in PBS (20 mm, pH 7.4) were used as an H2S source. Probes (1–
10 mm) were diluted in PBS (50 mm, pH 7.4). For the selectivity
experiment, biologically relevant compounds (100 mm) were prepared as stock solutions in PBS, then added to probe. The reaction
mixture was shaken thoroughly before emission spectra were recorded. For the time-course experiment, probe (1 mm) in PBS was
added to Na2S (100 mm) at room temperature, and the fluorescence intensity was measured at different time points. For the pHdependent experiment, 1 (1 mm) and Na2S (100 mm) were incubated in PBS at different pH values. The quantum yields of both
probes were tested in PBS buffer, with fluorescein (f= 0.95) and
rhodamine B (f= 0.69) as reference fluorophores.
Acknowledgements
This work was supported by the MOST (2010CB126102), NSFC
(21332004, 21402007, 81272356), the MOE Science and Technology Development Center (20130031120040), Tianjin Municipality
Science and Technology Commission (15JCYBJC50100).
Fluorescence microscope experiments: Cell culture: HEK-293
cells were cultured at 37 8C, CO2 (5 %) in high glucose DMEM
(GIBICO) supplemented with FBS (10 %), penicillin (100 U mL¢1),
streptomycin (100 mg mL¢1), and l-glutamine (4 mm). The cells
were maintained in exponential growth plase, and then seeded in
a glass-bottom 35 mm plate (~ 2 Õ 104 cells per well). Cells were
passaged every 2–3 days and used between passages 3 and 10.
Keywords: bioimaging · biosensors · fluorescent probes ·
hydrogen sulfide · thiolylsis of NBD amine
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Confocal imaging experiments: Cells were imaged on an FV1000
inverted fluorescence confocal microscope (Olympus, Japan) with
a UPLSAPO 20 Õ objective lens. All images were analyzed with
Olympus FV1000-ASW software. For live-cell imaging, cells were
first treated with probe at 37 8C for 30 min and then incubated
with Na2S (50 and 200 mm) for 1 h. Control cells were treated with
just probe. Emission was collected in the green channel (lex =
488 nm, lem = 500–600 nm) for 1, and in the red channel (lex =
546 nm, lem = 555–655 nm) for 2.
Co-localization: HEK293 cells were incubated with Na2S (100 mm)
for 30 min, washed with PBS, then incubated with Mito-Tracker
Green FM (0.25 mm) and 2 (2 mm) for 30 min. The medium was replaced by PBS, and cells were imaged via the green (lex = 488 nm,
lem = 500–530 nm) and red channels (lex = 546 nm, lem = 555–
655 nm).
Thiol stimulation experiments: HEK293 cells were co-incubated
with thiols d-Cys or l-Cys (100 mm) and probe (2 mm) for 1 h at
37 8C under CO2 (5 %). The medium was replaced by PBS, and cells
were imaged immediately. For inhibition experiments, cells were
pre-incubated with PPG (50 mg L¢1) for 20 min and then with thiols
(100 mm) and probe (2 mm) for 1 h without medium exchange.
After washing as described above, cells were imaged immediately.
H2O2 stimulation experiments: HEK293 cells were incubated with
1 (2 mm) for 30 min at 37 8C under CO2 (5 %), washed with PBS, and
then incubated with H2O2 (50, 150, or 200 mm) for 30 min. The cells
were imaged via the green channel (lex = 488 nm, lem = 500–
600 nm). HEK293 cells were incubated with H2O2 (200 mm) for
30 min at 37 8C under CO2 (5 %), washed with PBS, and then incubated with 2 (2 mm) for another 15 min. The cells were imaged via
the red channel (lex = 546 nm, lem = 555–655 nm) at 0, 1, 2, 3, 4, 5,
7, 9, 11, 13, and 15 min.
In vivo imaging experiments: All experimental procedures involving animals were in accordance with the Guide for the Care and
Use of Laboratory Animals (NIH publications nos. 80–23, revised
1996) and were performed according to the institutional ethical
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Manuscript received: January 31, 2016
Accepted article published: March 8, 2016
Final article published: April 15, 2016
968
Ó 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim