Fluorescent Quantification of Protein Persulfidation

Free H₂S was measured in cells using the SF₇-AM fluorescent probe [30 (link)] (Sigma-Aldrich). The probe was dissolved in anhydrous DMF at 5 mM and used at 5 μM in serum-free RPMI. Live-cell image acquisition was performed using a Nikon Ti2 spinning disk confocal microscope. Global protein persulfidation was assessed on VSMC grown on glass coverslips as previously described [9 (link)]. Cells were incubated for 20 min with 1 mM 4-Chloro-7-nitrobenzofurazan (NBF-Cl, Sigma-Aldrich) diluted in PBS. Then, cells were washed with PBS and fixed for 10 min in ice-cold methanol. Coverslips were rehydrated in PBS and incubated with 1mM NBF-Cl for 1 h at 37 °C. In parallel, a Daz2-Cy5.5 solution was prepared by mixing 1mM Daz-2, 1 mM alkyne Cy5.5, 2 mM copper(II)-TBTA, 4mM ascorbic acid and incubating overnight at RT, followed by quenching for 1h with 20mM EDTA. Fixed cells were further incubated at 37 °C for 1h in the Daz2-Cy5.5 solution. Finally, coverslips were washed 3 times in methanol and 2 times in PBS, mounted in Vectashield mounting medium with DAPI, and visualized with a 90i Nikon fluorescence microscope. Persulfidation was measured as the ratio of Daz2-Cy5.5 over NBF-Cl signal per cell by two independent experimenter blinded to the conditions using the Fiji (ImageJ 1.53t) software.