Celltiter glo luminescence kit

CCD8-Lu fibroblasts were treated with 2 μM rotenone, 100 μM antimycin A, 500 mM KCN, and 10 mM CCCP overnight. Intracellular ATP was measured using a CellTiter-Glo Luminescence Kit (Promega, Madison, WI, USA). The cells were added to 96-well white-bottomed plates, and the CellTiter-Glo Luminescence reagent was added. The plates were placed on a shaker for 2 min and incubated for 10 min. The luminescent signals were evaluated using a microplate reader (Synergy HTX, Biotek, CA, USA). Estimates of intracellular ROS and membrane potential were performed via flow cytometry. ROS levels were detected using CM-H2DCFDA (Invitrogen) in cells. The fluorescent signal was emitted at 519 nm when H2DCFDA was transformed into fluorescent 2′,7′-dichlorofluorescein via ROS. The cells were incubated with 5 μM CM-H2DCFDA at 37 °C for 30 min and washed with Dulbecco’s phosphate-buffered saline (DPBS) twice before measurements were taken. The membrane potential was detected using a fluorescent probe of mitochondrial membrane potential, namely tetramethylrhodamine (TMRE) (Invitrogen). The cells were incubated with 500 nM TMRE at room temperature for 10 min and washed with DPBS twice before measurement.