Hydrogen Sulfide Donor Preparation

GYY4137 [morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate] (Cayman Chemicals, USA) and NaHS (Alfa Aesar, USA) were used as sources of H₂S and were prepared according to the manufacturer’s instructions. While NaHS releases H₂S instantaneously, GYY4137 is a water soluble H₂S donor that releases H₂S slowly for weeks both in vitro and in vivo^{24 (link),27 (link)}, and more closely parallels the biological effect of endogenous H₂S. For H₂S exposure experiments, a 10 mM GYY4137 stock solution was prepared in water in a 5 ml tube and was immediately diluted to make a working stock and added to cultures to achieve the desired final concentration. Experimental control samples received time-spent 25 µM GYY4137 (referred to as spent GYY) that was previously aerated for at least 120 days. An NaHS stock solution (100 mM) was prepared by equilibrating phosphate-buffered saline (PBS), made anoxic by bubbling with argon, with NaHS in a septum-sealed glass syringe. In order to avoid any trace metals catalyzing the oxidation of H₂S, the metal chelator DTPA (diethylenetriaminepentaacetic acid) was added to the buffer to a final concentration of 50 µM prior to addition of NaHS. Photo-oxidation was prevented by wrapping the solution-containing syringe with aluminum foil. Despite these precautions, the presence of polysulfide in stock solutions cannot be completely excluded.