Annexin 5 fluorescein isothiocyanate fitc and propidium iodide double staining kit

The Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining kit (BD Biosciences, USA) was used to measure cardiomyocytes apoptosis by flow cytometry. Briefly, IH-treated cardiomyocytes were collected, washed with ice-cold phosphate buffered saline (PBS), and resuspended in 500 µL binding buffer. Cells were then incubated with Annexin V-FITC and PI (5 µL each) for 15 min at RT in the dark. Data were acquired with a flow cytometer (FACSCalibur, BD Biosciences, USA) within one hour, and analyzed with the FlowJo software (BD Biosciences, USA).

Puerarin was obtained from the National Institute for the Control of Pharmaceutical and Biologic Products (Beijing, China). Human recombinant IL-1β was purchased from Peprotech (Rocky Hill, NJ). Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY). Endothelial cell growth factor (ECGF) and calcein AM (C-AM) was purchased from Sigma (St. Louis, MO). The annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining kit was obtained from BD Biosciences (San Jose, CA). Caspase-3 substrate DEVD-AFC was purchased from R&D Systems (Minneapolis, MN). Dimethyl sulfoxide (DMSO), sodium bicarbonate, penicillin-streptomycin, trypsin, RNase A, polyvinylidene difluoride (PVDF) membrane, and enhanced chemiluminescence (ECL) assay kit were from Beyotime (Nantong, China). The following antibodies were used: vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1; Abcam, Cambridge, MA), Bax (Biomol, Hamburg, Germany), and Bcl-2 and caspase-3 (Oncogene, Cambridge, MA). Horseradish peroxidase (HRP) conjugated goat anti-mouse and anti-rabbit secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA).