Binding buffer

Cell apoptosis was detected using the annexin V-Alexa Fluor 647/propidium iodide double staining method. The adherent ACHN cells were digested and collected after drug treatment. The cells were washed and resuspended with 1X binding buffer (Yeasen Biotech Co., Ltd.). Annexin V-Alexa Fluor 647 and propidium iodide staining solution (Yeasen Biotech Co., Ltd.) were added. A total of 10,000 cells per sample were analyzed by flow cytometry (NovoCyte, 2060R, ACEA Bioscience, Inc.; Agilent) following the reaction at room temperature and avoiding light. The data were analyzed using FlowJo v10 (FlowJo LLC).

The cell apoptosis was evaluated by flow cytometry analysis using Annexin V-FITC/PI apoptosis analysis, according to the manufacturer's instructions (Shanghai Yeasen Biotechnology Co., Ltd.). Briefly, PC12 cells were exposed to Sev for 24 h, and harvested using trypsin. The harvested cells were resuspended in 1X binding buffer (Shanghai Yeasen Biotechnology Co., Ltd.) at a concentration of 1×10⁶ cells/ml. A total of 5 µl of Annexin V-FITC and 10 µl PI staining solution was subsequently added to the suspensions. The suspensions were vortexed gently, and 400 µl of 1X binding buffer was added and incubated for 5 min at room temperature in the dark. The fluorescence of FITC and PI was analyzed by flow cytometry. Both early apoptotic (Annexin V-FITC⁺/PI⁻) and late apoptotic (Annexin V-FITC⁺/PI⁺) cells were included in cell apoptosis determinations.

Cells of each experimental group (100,000 cells/well) were added into a 24-well plate and three duplicated wells were set in each group. They were incubated at 37°C with 5% CO₂ for 48 h. The cells were collected, Annexin V and propidium iodide [Yeasen Biotechnology (Shanghai) Co., Ltd.] were added and cells were incubated at room temperature in the dark for 15 min and suspended in 1X Binding Buffer [Yeasen Biotechnology (Shanghai) Co., Ltd.] according to the manufacturer's instructions. Apoptosis was then detected by flow cytometry.