Annexin 5 fluorescein isothiocyanate pi apoptosis detection

Cells, cultured at a density of 1×10⁵/assay, were trypsinized into single cell suspensions and fixed with 70% ethanol for 30 min on ice. Then, the cells were stained with propidium iodide (PI) using the CycleTEST TM PLUS DNA reagent kit (BD Biosciences, San Jose, CA, USA) and analyzed using a FACS Calibur flow cytometer (BD Biosciences) equipped with BD CellQuest software v6.1 (BD Biosciences). For apoptosis analysis, cultured cells were harvested by trypsinization and were double stained with Annexin V-fluorescein isothiocyanate/PI apoptosis detection (BD Biosciences) according to the manufacturer's protocol, and analyzed with the aforementioned flow cytometer and software.

Transfected cells were harvested by trypsinization, washed with cold PBS and fixed with 70% cold ethanol for 30 min at ice. For cell cycle analysis, fixed cells were washed and stained with propidium iodide (PI)/RNase A solution (BD Biosciences) in darkness for 30 min at 37˚C. Stained cells were applied for cell cycle distribution analysis using a FACSCalibur flow cytometer (BD Biosciences). For apoptosis analysis, fixed cells were stained with Annexin V-fluorescein isothiocyanate/PI apoptosis detection (BD Biosciences), followed by flow cytometry analysis according to the manufacturer's instructions. Data were analyzed using FlowJo^™ v.10.8 software (FlowJo, LLC).