Annexin 5 fluorescein isothiocyanate fitc propidium iodide apoptosis detection kit

Testosterone, bovine serum albumin (BSA), D-glucose, trypsin/EDTA solution and DMSO were purchased from Sigma-Aldrich; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The MTS CellTiter 96 Aqueous kit was purchased from Promega (Madison, WI, USA). The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit was obtained from BioVision, Inc. (Milpitas, CA, USA). Assay kits for the measurement of NO production, NOS activity, and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) concentrations were purchased from Jiancheng Bioengineering Research Institute (Nanjing, China). The tumor necrosis factor (TNF)-α concentration assay kit was obtained from R&D Systems (Minneapolis, MN, USA).

The rate of cell apoptosis was determined using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (BioVision, Inc., Milpitas, CA, USA). HRGECs were detached with trypsin-EDTA and washed with phosphate-buffered saline (PBS). They were subsequently resuspended in a binding buffer [10 mM HEPES (pH 7.4); 150 mM NaCl; 5 mM KCl; 1 mM MgCl₂; 1.8 mM CaCl₂] containing Annexin V-FITC (1 g/ml) and further incubated for 20 min at the room temperature. At 10 min prior to the end of incubation, PI (10 g/ml) was added to the cell suspension to stain necrotic cells. The cells were then analyzed via fluorescence-activated cell sorting using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with a 488 nm excitation laser, and analyzed using ModFit LT 2.0 software (Verity Software House, Topsham, ME, USA).