Jc 1 dyeing working solution

A2780/PTX cells seeded at a density of 1×10⁵ cells/well were incubated with different PTX formulations at equivalent PTX concentrations of 5 μM. The cells incubated with serum-free DMEM culture medium were served as negative control. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a chemical inhibitor of oxidative phosphorylation, could cause rapid mitochondrial membrane depolarization as the negative control. After 24 h incubation, cells were washed, collected and then mixed in 0.5 mL culture medium and 0.5 mL pre-prepared JC-1 dyeing working solution following the manufacturer's protocol (Beyotime, China) by flow cytometry (BD, USA). Additionally, MMP changes resulted from PTX formulations were also visualized by fluorescence image assay [31 ]. After incubation, cells were washed two times with cold PBS and stained with JC-1 dyeing working solution following the manufacturer's protocol (Beyotime, China) for 30 min. Fluorescence images were taken using a fluorescence microscopy (Olympus IX71, Tokyo, Japan) with a 60× objective.

MSCs were washed with PBS, and JC-1 dyeing working solution (Beyotime) mixed with fresh medium was added. The cells were incubated at 37°C for 20 min in a cell incubator. After incubation at 37°C, the supernatant was aspirated and the cells were washed twice with JC-1 staining buffer (Beyotime). Afterward, 2 ml of cell culture medium was added, and the results were observed under a fluorescence microscope. Relative degrees of mitochondrial polarization were quantified by measuring the red-shifted JC-1 aggregates and the green-shifted monomers.