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

Lack of reliable methods to accurately measure hydrogen sulfide (H ₂S) 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 H ₂S 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.