Tetramethylrhodamine ethyl ester (TMRE) was used to analyze changes of mitochondrial membrane potential. Specifically, A549 cells were seeded into a 12-well plate and treated with CLP nanoparticles at various concentrations for 12 hours or at 65 μM for predetermined time periods. Then, cells were incubated with 100 nM TMRE for 30 min. The incubated cells were washed twice with PBS, collected, and subsequently assayed by flow cytometry, with FCCP (carbonyl cyanide p-trifluoromethoxy phenylhydrazone) used as a positive control.

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