Mito tracker

Indicated HEI-OC1 cells (2 × 10⁵ cells per well) were seeded into 6-well plates and grown to 80% confluency, and then cells were treated with or without 80 µM t-BHP for 4 h. After treatment, HEI-OC1 cells were cultured with fluorescent Mito-Tracker (a hydrophobic compound, easily permeates intact live cells and becomes trapped in mitochondria after it gets into cells) (Abcam, United States) for 1 h in a 33°C 10% CO₂ humidified incubator. At the end of incubation, the cells were digested and kept on ice protected from light. The fluorescence arising from Mito-Tracker (Texas Red channel) was then detected by a BD FACSAria II instrument (BD, United States). A total of 20,000 events were collected per sample, and polygonal gating was used to exclude debris. Data were analyzed with FlowJo Software. All experiments were performed with five replicates.

Cells were incubated 37 °C, 95% air and 5% of CO2, for 30 min with 5 nM Mito Tracker (Rosamine-based Mitotracker dye-Invitrogen) to identify mitochondria, 5 μ M (Red Mitochondrial Superoxide Indicator) MitoSOX to determine Ros mitochondria production, 100 n M TMRE (Tetramethylrhodamine, Ethyl, Ester) to identify mitochondrial potential membrane. Then Mito Tracker, Mitosox, and TMRE stained and unstained control cells were analyzed by flow cytometry (FACS Calibur, BD Biosciences) followed by analysis of mean fluorescence intensity of 10,000 events by Cellquest software (BD Biosciences).