Novel Applications of Lead Acetate and Flow Cytometry Methods for Detection of Sulfur-Containing Molecules

Abstract

Hydrogen sulfide (H2S) is the most recently established gaseous vasodilator, enzymatically produced from cysteine metabolism, involved in a number of pathophysiological processes. However, its accurate detection in vivo is critical due to its volatility and tendency to form sulfane sulfur derivatives, thus limiting the data interpretation of its biological roles. We developed new applications of the simple and rapid method to measure H2S release in cell culture systems, based on the lead acetate strip test. This test, previously prevalently used in microbiology, was compared with the agar trap method, applied, in parallel, on both cell cultures and cell-free samples. Sulfane sulfur represents the major species derived from intracellular H2S. Various fluorescent probes are available for quantitation of H2S derivatives intracellularly. We present here an alternative to the classic imaging method for sulfane sulfur evaluation, running on a flow cytometer, based on SSP4 probe labeling. Flow cytometry turned out to be more direct, fully quantitative and less time-consuming compared to microscopy and more precise with respect to the fluorescence multi-plate reader assay. The new application methods for H2S determination appear to be fully suitable for the analysis of H2S release and sulfane sulfur content in biological samples.

Keywords

• H₂S
• SSP4
• fluorescent probe
• sulfane sulfur species
• lead acetate test strips
• flow cytometer
• fluorescence imaging
• agar trap
• cell culture