3.9. Mitochondrial Membrane Potential Assay

The potential of apoptosis due to mito stress was analyzed by JC-1 Mitochondrial membrane potential assay kit (JC-1 MitoMP, Dojindo Inc., Kumamoto, Japan). HSC-3 cells were cultured in standard cell culture condition with DMEM medium with 10% FBS and treated with or without OI extract (50 ug/mL) and cisplatin (5 ng/mL) for different time incubation periods of 0, 50, 100, 150 and 200 min in a 96-well culture vessel. After the incubation period, the medium was discarded and cells were washed twice with Hanks HEPES buffer, and cells were lysed with RIPA buffer and relative fluorescent unit (RFU) was noted with Ex 525 nm/Em 545 nm using a multimodal spectrofluorometer (Multiskan FC, Thermo Fisher Scientific Inc., Pittsburgh, PA, USA). Values were expressed in RFU vs. time of test compound incubation.