Annexin 5 fluorescein isothiocyanate fitc propidium iodide staining kit

An Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Staining kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect apoptotic cells. Cells were harvested 48 h following transfection and resuspended in 1X binding buffer at a concentration of 1×10⁶ cells/ml. Subsequently, cells were stained with 5 µl Annexin V-FITC and 5 µl PI for 15 min at room temperature in the dark. Cells were acquired using a BD Accurri^™ C6 flow cytometer (BD Biosciences) and data was analyzed using FlowJo software version 7.6.2 (Tree Star, Inc., Ashland, OR USA).

DU145 and LNCaP cells were cultured in RPMI 1640 medium (Gibco) with 2 Gy IR treatment. Annexin V-Fluorescein isothiocyanate (FITC)/Propidium iodide (PI) staining kit (BD Biosciences) was used to detect the apoptosis cells numbers of PCa cells following the manufacturer’s protocol. The result was determined by BD FACS Canto™ II flow cytometer (BD Biosciences).

The effects of metformin on ovarian cancer cell apoptosis were assessed by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining kit (BD Pharmingen; BD Biosciences, San Jose, CA, USA). As cells transduced with lenti-virus carried green fluorescence, their apoptosis rates were assessed by using Annexin V-phycoerythrin (PE)/7-aminoactinomycin D (7-AAD) staining kit (BD Pharmingen; BD Biosciences). After 24 h of exposure to metformin, cells were washed twice with ice-cold PBS, re-suspended in 500 µl binding buffer, and stained with 5 µl FITC-conjugated 5 µl Annexin V or 5 µl Annexin V-PE conjugated and 7 µl 7-AAD, following gentle mixing, cells were incubated at room temperature shielded from light for 15 min. Following washing with binding buffer, flow cytometry analysis was performed using a flow cytometry sorting system (MoFlo XDP) with Summit 6.2 software (both from Beckman Coulter, Inc., Brea, CA, USA). Each assay was run at least three times.