Sf7am

H₂S levels were determined by a fluorometric assay in cell lysate and supernatant of BAEC that have undergone the different protocol treatments by using the specific fluorescent H₂S probe SF7AM (Ex. 475 nm, Em. 500–550 nm, Tocris, UK). The volume of samples used was 50 μl for the supernatant, while cell lysate was used at a concentration of 0.5 mg/ml (50 μl). The samples, standards (Na₂S) and blanks were pipetted into a black flat-bottomed 96-well plate by using RIPA solution as a dilution buffer. SF7AM was used at a final concentration of 10 μM and the total volume for each well was 200 μl. The plate was read at 37 °C throughout 90 min incubation period under orbital shaking conditions. Total H₂S levels were quantified against a calibration curve obtained with Na₂S (50nM-250μM). In another set of experiments, we measured live H₂S production in cells. BAEC were incubated in NG or HG environment for 30 min and thereafter SF7AM (2.5 μM) was added for 30 min [56 (link)]. Following incubation, the medium was replaced to eliminate SF7AM excess and the cells were treated according to the different protocols described (AP123 10 nM or AP123 + PAG) for 2 h. Fluorescence readings were carried out straight after AP123 addition for 2 h at 30-min intervals (supplemental method M2).