Annexin 5 fluorescein isothiocyanate propidium iodide apoptosis detection kit

Cells were plated at a density of 2×105 cells/2 ml medium on 6-well plates for 24 h. Following treatment with various concentrations of FKA (0, 5, 10 and 30 µM) for 24 h, cell apoptosis was detected using DAPI staining. Cells were fixed with 90% ethanol/5% acetic acid for 1 h at room temperature. Following 2 washes with PBS, cells were incubated with DAPI solution (1.5 mg/ml in PBS) for 30 min at room temperature. Images of DAPI fluorescence were captured using a fluorescence microscope (magnification, ×200; Nikon Corporation). After treated by different concentrations of FKA (0, 5, 10 and 30 µM) for 24 h at 37°C, cells were digested with trypsin and centrifuged at 120 × g for 5 min at 4°C. Following 2 washes with PBS, levels of apoptosis were analyzed using an Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit (BD Biosciences), according to the manufacturer's protocol. Quantification of fluorescence was determined using flow cytometry (FACSCalibur™; BD Biosciences), and the data were analyzed using WinMDI software v2.8 (Purdue University Cytometry Laboratories).

PTX and FKA were purchased from Sigma (St. Louis, MO, USA). Sodium aescinate (Aes) (≥98%) was purchased from Meilunbio^® (Dalian, China). Monoclonal rabbit anti-human P-gp (cat. no. 13978) was purchased from Cell Signaling Technology (MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Annexin V‑fluorescein isothiocyanate/propidium iodide Apoptosis Detection Kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The cell cycle detection kit was purchased from Sigma (St. Louis, MO, USA). Dialysis membranes (MWCO 1000) were obtained from the Shanghai Medical Chemical Reagent Co., Ltd. (Shanghai, China). Phosphate buffer and 0.25% trypsin were purchased from Gibco, Invitrogen Corp (Ontario, USA).