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.

1 × 10⁵ cells were cultured on 14-mm cell climbing slices. Cells at 80% confluency were incubated with a working solution of Mito-Tracker (Abcam, USA) for 1 h at 37°C. Cell climbing slices were then fixed, permeabilized, and sealed with mounting medium containing DAPI (Abcam, USA). Image acquisition was performed using the TCS SP8 confocal laser scanning microscopy (Leica, Italy).

After treatments, cells were loaded with Mito tracker (Cayman Chemical, Ann Arbor, MI, USA) in PBS for 30 min at 37 °C, washed with PBS and then the cells were fixed with methanol for 10 min at −20 °C and blocking in 5% BSA in PBST for 30 min. Fixed cells were incubated overnight at 4 °C with primary antibody nephrin (ab216341, Abcam, Cambridge, MA, USA), cytochrome c (12963S, Cell Signaling, Beverly, MA) or TRPC6 (ab62461, Abcam, Cambridge, MA, USA) diluted in PBST and then washed three times in PBST and incubated for 1 h at room temperature with anti-rabbit or anti-mouse Alexa Fluor 488 or 594 (A-11059 and A27027, Molecular Probes). The mitochondria were labeled with Mito tracker and the nuclei were visualized by DAPI (ab228549, Abcam, Cambridge, MA, USA) staining. Images were taken using LEICA laser scan microscope.