Mitochondrial membrane potential (MMP) assay

Fluorescence of Rhodamine-123 was used to monitor changes in MMP. Loss of Rhodamine-123 from the mitochondria decreases the intracellular fluorescence intensity during cell apoptosis due to the depolarization of MMP. In brief, Hs 683 cells were treated with TA for 48 h at a range of concentrations (0, 1, 5 and 10 µM). Rhodamin-123 was added 30 min prior to the termination of the experiment, and incubated at 37°C for 30 min. Cells were centrifuged at 400 × g for 5 min at 20°C and then washed three times with phosphate buffered saline (PBS). Fluorescent intensity was measured at an excitation wavelength of 488 nm and emission wavelength of 529 nm using a fluorescence microplate reader. The fluorescence of each TA-treated concentration group was compared with an untreated group in three independent experiments.