MitoTracker Red CMXRos (YEASEN, 40741ES50) and DCFH-DA (Beyotime, s0033) were used to determine the mitochondrial membrane potential and to analyze mitochondrial ROS production, respectively. After trypsinization, HEI-OC1 cells were collected by centrifugation and washed with cold 1× PBS. The cell mass was resuspended in a solution containing MitoTracker Red CMXRos or DCFH-DA for 15 min at room temperature in the dark, followed by washing with cold PBS and analysis by flow cytometry (BD FACSCalibur).
FITC Annexin V (BD, 556547) was used for the analysis of apoptosis, and propidium iodide (PI) was used to distinguish viable cells from nonviable cells. HEI-OC1 cells were trypsinized for 5 min and collected by centrifugation at 1,000 rpm for 5 min, washed with cold 1× PBS, resuspended in 1× binding buffer, and aliquoted at 1 × 105 cells (100 μl) into a 5 ml culture tube. FITC Annexin V and PI were added to the tube, gently vortexed, incubated for 15 min at room temperature in the dark, and analyzed by flow cytometry within 1 h.
FITC Annexin V (BD, 556547) was used for the analysis of apoptosis, and propidium iodide (PI) was used to distinguish viable cells from nonviable cells. HEI-OC1 cells were trypsinized for 5 min and collected by centrifugation at 1,000 rpm for 5 min, washed with cold 1× PBS, resuspended in 1× binding buffer, and aliquoted at 1 × 105 cells (100 μl) into a 5 ml culture tube. FITC Annexin V and PI were added to the tube, gently vortexed, incubated for 15 min at room temperature in the dark, and analyzed by flow cytometry within 1 h.