2.6. H2S concentration measurement

H₂S concentration was measured as described previously [23]. Briefly, 500 μl of culture medium from different treated cells was mixed with 250 μl of 1% zinc acetate in a test tube. Subsequently, N-dimethyl-p-phenylenediamine sulfate (20 mM, 133 μl) in 7.2 mM HCl and FeCl₃ (133 μl) in 1.2 mM HCl were added to this test tube and incubated for 10 min at room temperature. To remove the protein in the culture, trichloroacetic acid (10% w/v, 250 μl) was added to the reaction, and the protein was pelleted by centrifugation at 12,000 rpm for 5 min. The absorbance (670 nm) intensity was measured by a spectrophotometer (M1000, TECAN, Austria GmbH, Austria). The H₂S concentrations of each sample were calculated against a calibration curve of NaHS (3.125–250 μM).