Enhanced detection of hydrogen sulfide generated in cell culture using an agar trap method

Lack of reliable methods to accurately measure hydrogen sulfide (H2S) produced in vitro has impeded research on the physiology of this gaseous mediator. Current in vitro methods involve measurement of H2S in cell culture media following incubation with H2S-releasing compounds. However, this method is inaccurate because H2S gas has a short life and thus evades detection. To overcome this, we have adapted a method that employs a modified agar layer to instantly trap H2S, allowing measurement of H2S accumulated with time. The amount of H2S trapped in the agar is quantified using an in situ methylene blue assay. We were able to detect H2S produced from sodium hydrogen sulfide (NaHS) added at concentrations as low as 10 μM. Following a 24-h incubation of endothelial-like or vascular smooth muscle cells with 50 μM NaHS, we were able to recover twice more H2S than conventional methods. When H2S-releasing compounds l-cysteine and N-acetylcysteine were added to the cell culture, the amount of H2S increased in a concentration-, time-, and cell line-dependent manner. In conclusion, we have developed an improved method to quantify H2S generated in vitro. This method could be used to screen compounds to identify potential H2S donors and inhibitors for therapeutic use.