Quantifying Tissue H2S Production

H₂S production capacity was measured by using the lead acetate/lead sulfide method (5 (link), 24 ). Briefly, brain, liver, or kidney tissue was first placed in 1.5 mL microcentrifuge tubes containing 250 μL of 1 × passive lysis buffer (Promega) and homogenized, followed by multiple rounds of flash freezing/thawing by using liquid nitrogen. After homogenization and lysis, protein concentration was measured with bicinchoninic acid assay (BCA) kit (Bio-Rad) followed by normalization of proteins via additional 1 × passive lysis buffer. Next, the lead acetate/lead sulfide assay was set up by initially preparing the reaction mixture of 10 mMl-cysteine (No. 168149; Sigma) and 1 mM pyridoxal phosphate (PLP; No. 9255; Sigma) in phosphate buffered saline (PBS), with 150 μL placed into each well of a 96-well plate. One hundred micrograms of protein from each tissue or 20 μL of plasma was added to each respective well. Then, the plate was overlaid with lead acetate embedded filter paper and incubated at 37°C until lead sulfide was detected for quantification by using ImageJ densitometry analysis via the IntDen function after subtracting background levels obtained from reaction mixture-only wells with no tissue or protein added.