Annexin 5 fitc pi apoptosis assay kit

Apoptosis was determined using the Annexin V-FITC/PI apoptosis assay kit (Sungene Biotech, China). Flow cytometry with a BD FACSVantage SE cytometer was used to detect the apoptosis rate of cells. Annexin V-positive/PI-negative podocytes were considered to indicate the early stages of apoptosis, whereas annexin V-positive/PI-positive podocytes were considered to be late apoptotic or necrotic cells.

Cytotoxicity was estimated by a Cell Counting Kit-8 (CCK-8) colorimetric assay (Sigma-Aldrich, St. Louis, MO, United States). HK-2 cells were seeded onto a 96-well plate and treated with various concentrations of BSA or PA (Sigma, United States, P9767) (0, 100, 150, 200, 250, 300, 350, 400, 450 μM) for 24 h. Then, the treated cells were incubated with a CCK-8 kit (100 μL/well) for 2 h at 37°C in a 5% CO2 incubator. After the plate was agitated, the absorbances of each well at 450 nm were measured using a multiwell fluorescence plate reader (Thermo Scientific Varioskan Flash).
To evaluate the extent of cell apoptosis, an Annexin V-FITC/PI apoptosis assay kit (Sungene Biotech, Tianjin, China) was used to assess cell apoptosis. Briefly, after the indicated treatments, the cells were stained with 10 μg/ml Annexin V/FITC for 30 min at 37°C and with 5 μg/ml propidium iodide (PI) for 5 min at room temperature, followed by flow cytometry analysis using a BD FACSVantage SE cytometer.