Jc 1 mmp assay kit

DCVC-stimulated changes in the MMP were assessed using the fluorescent reagent JC-1 with the JC-1 MMP assay kit (Cayman Chemical) following the manufacturer's protocol. JC-1 is a potential-sensitive lipophilic dye that selectively accumulates in mitochondria and changes fluorescence from red in cells with healthy MMP to green with mitochondrial membrane depolarization. HTR-8/SVneo cells were seeded at a density of 50 000 cells per well and cultured for 24 h in a black, clear-bottom 96-well plate. Cells were treated with 5, 10, or 20 μM DCVC for 5, 10, and 24 h. Following treatment, cells were washed once with 200 μl of HBSS and then incubated with JC-1 dye in HBSS for 30 min at 37°C. Then, JC-1 dye was removed, cells were washed once with HBSS, 200 μl of fresh HBSS was added to each well, and fluorescence was measured using a SpectraMax M2e Multi-Mode microplate reader (Molecular Devices).
In separate experiments, JC-1 fluorescence was visualized by epifluorescence microscopy. Cells were grown at a density of 400 000 cells per well in a 6-well plate. Following incubation with 5, 10, or 20 μM DCVC for 10 or 24 h, JC-1 was added to each well, and plates were incubated at 37°C for 30 min. Cells were then washed, and fresh HBSS was added back to the wells. Cells were viewed using an EVOS digital inverted fluorescence microscope.