Analyzing Cell Cycle and Apoptosis by Flow Cytometry

Consistent with the previous description, cell cycle distribution, and apoptosis were detected by flow cytometry [24 (link), 36 (link), 37 (link)]. For cell cycle assay, after transfected with Importin-7 siRNAs, cells were digested and washed twice with 4 °C PBS and fixed with a mixture containing 2 ml cold 70% ethanol solution and 500 μl PBS at 4 °C overnight. After being washed with PBS, cells were prepared into a single cell suspension using a Propidium Iodide (PI)-containing cell cycle detection kit (KGA512, KeyGEN BioTECH, Nanjing, China) according to the instruction and left to stand in the dark for 30 min. Three independent tests for cell cycle distributions of each group were ultimately examined with flow cytometry.
For apoptosis assay, cells were treated according to the indicated conditions, then collected and washed twice using 4 °C PBS. An Annexin V-FITC/PI-containing apoptosis detection kit (KGA108, KeyGEN BioTECH, Nanjing, China) was used to stain the apoptotic cells according to the instruction. Flow cytometry was performed to determine the apoptotic rate from three independent experiments.