Annexin 5 fitc binding buffer

Cultured cells were suspended in PBS and counted. Resuspended cells (5–10×10⁴) were centrifuged at 1,000 rpm for 5 minutes and resuspended in Annexin V-FITC binding buffer (195 μL) (BD Pharmingen, Franklin Lakes, NJ, USA). Annexin V-FITC (195 μL) was supplemented and the cells were incubated away from light at 20°C–25°C for 10 minutes. PI (10 μL) was further added, followed by incubation in the darkness. Apoptosis was quantified by using flow cytometry. CellQuestPro software (BD Biosciences, Franklin Lakes, NJ, USA) was used for analyzing apoptosis condition.

Following culture for 12, 24 or 48 h, apoptosis was detected using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Cells in 6-well plates (5×10⁵/well) were detached by trypsinization and washed three times in phosphate-buffered saline, centrifuged at 4°C at 1,000 × g for 5 min and resuspended in 195 µl Annexin V-FITC binding buffer (BD Biosciences). Annexin V-FITC (5 µl) was added and mixed. Following this, the U2OS and MG63 cells were stained by Annexin V-FITC in binding buffer in the dark for 10 min at room temperature. The cells were subsequently centrifuged at 4°C at 1,000 × g for 5 min and were resuspended in 190 µl Annexin V-FITC binding buffer. Finally, 10 µl propidium iodide (PI) staining solution was added and mixed at 4°C for 15 min. The U2OS and MG63 cells were maintained on ice in the dark and immediately subjected to flow cytometric analysis (12 ). The data were analyzed using the Cell Quest software (version 7.5.3; BD Biosciences, San Jose, CA, USA).