Complex 4 rodent enzyme activity microplate assay kit

A TMRE-Mitochondrial Membrane Potential Assay Kit (Abcam, Cambridge, UK) was used to determine the mitochondrial membrane potential (MMP) in the C28/I2 cells. C28/ I2 cells (3 x 10 5 ) were incubated with the MMP-sensitive fluorescent TMRE for 30 minutes at 37°C (1000 nM FCCP was added to the positive control cells 10 min prior of TMRE). Cells were then trypsinized, centrifuged, and resuspended in 0.4 mL Dulbecco's phosphatebuffered saline with 0.2% bovine serum albumin and analyzed for TMRE fluorescence using the fluorescence spectrophotometer (Hitachi, Tokyo, Japan) with an excitation wavelength of 575 nm and an emission wavelength of 549 nm. Complex IV activity was measured using a Complex IV Rodent Enzyme Activity Microplate Assay Kit (Abcam, Cambridge, UK) according to the manufacturer instructions. In brief, C28/I2 cells were collected and lysed with the extraction detergent. The protein samples were then serially diluted and incubated at room temperature for 3 hon the plate, in each well of which the enzyme-linked monoclonal antibody against complex IV has immobilized. The binding was then assayed using the fluorescence spectrophotometer (Hitachi, Tokyo, Japan) at an excitation wavelength of 575 nm and an emission wavelength of 549 nm. The results are reported as a percent level of the control.