2.3. H2S Fluorescence Measurement

We monitored the H₂S release and intracellular uptake from P* with the H₂S fluorescent probe 3-oxo-3H-spiro-[isobenzofuran-1,9’-xanthene]-3’,6’-diyl-bis(2-(pyridin-2-yl-di-sulfanyl)-benzoate), (WSP-5) in the ATDC5 cells. The ATDC5 cells were grown on glass coverslips in a 12-well plate (8 × 10⁴ cells/well) for 24 h. The cells were washed once with FBS-free DMEM/F12 and incubated with 10 µM WSP-5 (in FBS-free DMEM/F12 + 100 µM hexadecyl (trimethyl)ammonium bromide) for 30 min. After washing the cells with phosphate-buffered saline (PBS), they were incubated with 1 mM P* (in FBS-free DMEM/F12 + 100 µM hexadecyl (trimethyl)ammonium bromide) for 30 min. Afterwards, fluorescence imaging was performed at room temperature using a Zeiss Axiovert 200 equipped with a xenon lamp, polychromator, Chroma filters (exciter: ET470/40; emitter: ET525/50; beamsplitter: T495lp), and a CoolSNAP fx-HQ CCD-camera (Visitron Systems GmbH, Puchheim, Germany). For each condition, 8 images were taken randomly and analyzed using ImageJ (https://imagej.nih.gov/ij/, accessed in 2015). For background correction, fluorescence was measured in cell-free areas.