Measuring Intracellular Sulfane Sulfur

Vascular endothelial cells were cultured on glass-bottom dishes until confluence and were treated with TGF-β₁ for 24 h. After washing with Ca²⁺- and Mg²⁺-free phosphate-buffered saline (CMF-PBS, Nissui Pharmaceutical), vascular endothelial cells were fixed using 4% paraformaldehyde in phosphate-buffered solution (Nacalai Tesque) at room temperature for 25 min. After washing twice with CMF-PBS, vascular endothelial cells were stained with fluorescent working solution containing 20 µmol/L SSP4 (Dojindo, Kumamoto, Japan) and 0.5 mmol/L cetyltrimethylammonium bromide (Nacalai Tesque) in serum-free DMEM at 37 °C for 1 h. After washing with CMF-PBS, serum- and phenol red-free DMEM and Fluoro-KEEPER Antifade Reagent (Nacalai Tesque) were added. Fluorescence images were captured using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) at λex = 482 nm and λem = 515 nm to measure the content of sulfane sulfur as an indicator of RSS. Intracellular low-molecular-mass RSS were quantified as described previously [49 (link)] using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) with β-(4-hydroxyphenyl)ethyl iodoacetamide (Molecular Biosciences, Boulder, CO, USA).