Measuring Apoptosis in Transfected Cells

Cells transiently transfected with siRNA or miRNA were plated in 6-well plates with or without siRNA-100 transfected, then the PV at different titers was added at 24h post-trasfection. After post-infection for 24 h, the cells’ apoptosis was measured by ApopNexin^™ FITC Apoptosis Detection Kit (APT750, Millipore, Temecula, CA) as we previously described44 (link). Briefly, cells were harvested and spun down (400 g, 5 min) and washed by PBS, resuspending cells in 1× bind buffer at a concentration of 10⁶ cells/ml. Then, took 200 μl cell suspension and added 3 μl of the annexin conjugate ApopNexin™ FITC and 2 μl PI. Finally, samples were mixed and incubated for 15 min at room temperature in the dark and placed the cells on ice. Fluorescence due to FITC and PI staining was measured in a flow cytometer (Cytomics FC 500, Beckman Coulter).