4.10. Measurement and Staining of Mitochondrial Superoxide (MitoSOX)

ImKCs (5 × 10⁴ cells/mL) and Hep3B cells (3 × 10⁴ cells/mL) were plated, and 10% FBS (fetal bovine serum) was added to Dulbecco’s Modified Eagle’s Medium (DMEM) at 37 °C for 24 h. After 24 h, ImKCs were treated with ZC and Mito-TEMPO for 6 h in a medium without FBS (fetal bovine serum) added, with or without LPS. In Hep3B cells, ZC and Mito-TEMPO were treated for 24 h in a medium without fetal bovine serum (FBS) added, with or without TNF-α. Cells were washed twice with phosphate-buffered saline (PBS) and stained with MitoSOX (Invitrogen, Waltham, MA, USA, {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008) reagent for 15 min. The cell was washed away with PBS, and the fluorescent expression value was measured using a microplate reader (TECAN, Spark). For cell staining, cells were cultured under the same conditions, stained with MitoSOX (Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008) reagent for 15 min, washed with PBS, and fixed with 4% Paraformaldehyde (biosesang, PC2031-100-00) for 20 min. It was washed with PBS and stained with DAPI (diamidino-2-phenylindole) for 5 min. For the quantitative analysis of mtROS production, the scanned cells were captured using a DMi8 (Leica Camera, Wetzlar, Germany) at 400× magnification.