Iron chloride fecl3

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). Ferric ammonium citrate (FAC) and iron chloride (FeCl₃) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Baicalein, erastin, ferrostatin-1 (Fer-1), and liproxstatin-1 (Lipo-1) were purchased form Selleck Chemicals (Houston, TX, USA).

H₂S production was evaluated as reported by d’Emmanuele di Villa Bianca and coworkers35 . Human urothelial samples or T24 cells were homogenized with lysis buffer containing potassium phosphate buffer (100 mM, pH 7.4), sodium orthovanadate (10 mM) and proteases inhibitors. Homogenates were added for 40 min in a reaction mixture (total volume 500 μl) containing piridoxal-5′-phosphate (2 mM, 20 μl) and saline (20 μl) or L-cysteine (10 mM, 20 μl, Sigma-Aldrich, Milan, Italy) to measure enzyme activity i.e. H₂S generation. The reaction was performed in parafilmed Eppendorf tubes and initiated by transferring tubes from ice to a water bath at 37 °C and carried out for 40 min. After incubation, zinc acetate (1%, 250 μl, Sigma-Aldrich) and trichloroacetic acid (10%, 250 μl) were added. Subsequently, N,N-dimethyl-p-phenylendiamine sulfate (DPD, 20 mM, Sigma-Aldrich) in HCl (7.2 M) and iron chloride (FeCl3, 30 mM, Sigma-Aldrich) in HCl (1.2 M) were added and optical absorbance of the solutions was measured after 20 min at a wavelength of 667 nm. All samples were assayed in duplicate and H₂S concentration was calculated against a calibration curve of NaHS (3.9–250 μM, Sigma-Aldrich). Results were calculated as nmol/mg protein/min and expressed as mean ± SE. Data were analyzed by using one way ANOVA following by Bonferroni as post test. p < 0.05 was considered significant.