Flow cytometry for cell apoptosis

H9C2 cells were seeded on 6 cm plates at a density of 2 × 10⁶. After transfection and/or LPS stimulation for the indicated time, the apoptosis rate was determined with an annexin V/propidium iodide apoptotic kit (Beyotime, Jiangsu, P.R. China) according to the manufacturer’s instructions.¹⁴ Briefly, H9C2 cells were collected and rinsed twice with cold PBS. The washed cells were re-suspended in binding buffer. Then, FITC-Annexin V and propidium iodide were added to the buffer for 10 min in dark incubation at 25°C. After staining, the cell mixture was loaded in a flow cytometry system. The apoptosis rate was calculated as a sum of late apoptosis (upper-right quadrant) and early apoptosis (down-right quadrant).