Regents
The hydrogen sulfide fluorescent probe was provided by Professor Baocun Zhu (School of Water Conservancy and Environment, University of Jinan, Jinan, China). 1 mg probe was dissolved in 100 μL dichloromethane, then was diluted with DMSO (Sigma-aldrich, USA) to a final concentration of 1 mM. α-MEM was used to dilute the mother liquor to get different concentrations. The test concentration was 10 μM and the experiment was carried out at room temperature (25 ℃).
DL-propargylglycine (PAG) (cystathionine γ-lyase inhibitor, Sigma-Aldrich), Cysteine (Cys), NaHS, lipopolysaccharide (LPS) (Sigma-aldrich, USA), cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Tokyo, Japan).
MC3T3-E1 cell culture
The murine calvaria-derived MC3T3-E1 osteoblast-like cell line (Procell CL-0378, subclone 14) was provided by Procell Life Science and Technology CO., Ltd. Cells were seeded at 5 × 104 cells/mL into 25 cm2 flasks and maintained in α-MEM, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were maintained in an incubator containing a 5% carbon dioxide/air environment at 37℃.
Toxicity analysis
The influence of the H2S probe on MC3T3-E1 cell was examined by CCK-8. Briefly, MC3T3-E1 cells, seeded at a density of 5 × 104 cells·/ml on a 96-well plate, were maintained at 37 ℃ in a 5% CO2, 95% air incubator for 24 h. Then the cells were incubated with different concentrations (0, 5, 10, 20, 25, 37.5, 50, 75 and 100 μM) of probe suspended in culture medium for 24 h. Same as the probe group, the other plate of cells was incubated with same concentrations (0, 5, 10, 20, 25, 37.5, 50, 75 and 100 μM) of solvent. Subsequently, CCK-8 solution was added into each well for 2 h. The absorbance at 450 nm was then measured.
Application of H2S probe to access exogenous H2S levels
The cells were pre-treated with NaHS (50, 100, 150, 500 μM) for 30 min, then, treated with the H2S probe (10 μM) for 30 min. Fluorescence and bright field images were collected after PBS washing for three times. Green fluorescence was observed under the confocal microscope at excitation wavelengths of 405 nm. In order to control exposure, Smart Gain was kept at the same voltage in every photographs.
Application of H2S probe to access endogenous H2S levels
In the periodontium of mammalian host, H2S is produced using Cys mainly by CSE and CBS. The cells were pre-treated with Cys (100 μM, 200 μM) for 30 min, then, treated with the H2S probe (10 μM) for 30 min. Fluorescence and bright field images were collected after PBS washing for three times. Green fluorescence was observed under the confocal microscope at excitation wavelengths of 405 nm. In order to control exposure, Smart Gain was kept at the same voltage in every photographs.
PAG is an irreversible inhibitor of CSE. It can block the produce of endogenous H2S in MC3T3-E1. Therefore, we pre-treated cells with 50 μM PAG, 30 min, then cells were treated with or without Cys for 30 min. Last, fluorescence was examined as before, Smart Gain was kept at the same voltage in every photographs.
Addition of lipopolysaccharide (LPS) for inducing inflammation and assessment with H2S probe: The cells were incubated with 1, 2 μg/mL LPS for one day. Subsequently, the culture dish was washed with PBS for three times and incubated with 10 μM probe for 30 min. Then, the cells were washed with PBS, then the fluorescence imaging was examined by confocal microscope, Smart Gain was kept at the same voltage in every photographs.
