Fitc labeled annexin 5 pi staining

Flow cytometric analysis was performed to determine the presence of cell cycle arrest and apoptotic cells. After treatment with PA for 24 h, the cells were collected by trypsinisation and washed twice with PBS, fixed in ice-cold 80% ethanol, and stored overnight at 4°C. For analysis, the cells were washed twice with PBS and 10 mg/ml RNase A was added. Propidium iodide was added to the tubes at a final concentration of 0.05 mg/ml and incubated at 4°C for 20 min in the dark. Cell cycle analysis was performed with FACSCalibur flow cytometer (BD Biosciences, CA). FITC-labeled Annexin V/PI staining was performed according to the manufacturer’s instructions (Keygen, Nanjing, China). Briefly, 1 × 10⁶ cells/well were suspended in buffer containing FITC-conjugated Annexin V/PI at appropriate concentrations. The samples were analyzed by flow cytometry and 20,000 events from each sample were obtained to ensure adequate data.

The apoptosis rate of hippocampal neurons was carried out by flow cytometric analysis. Both attached and floating hippocampal neurons were harvested and washed twice with PBS, fixed in ice-cold 80% ethanol, and stored overnight at 4°C. Then, the cells were washed twice with PBS and 10 mg/mL RNase A was added. FITC-labeled Annexin V/PI staining was performed according to the manufacturer's instructions (Keygen, Nanjing, China). For each experiment, 20,000 cells were analyzed using Elite Flow Cytometry (BD Biosciences, San Jose, CA) and Cell Quest software (BD Biosciences). Triplicates were performed in all cases for the detection of early apoptotic cells.