FITC Annexin V Apoptosis Detection Kit I

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	Add 5 µl of FITC Annexin V and 5 µl PI and incubate or 15 min at room temperature (25°C) in the dark

Protocol tips
Add 5 µl of FITC Annexin V and 5 µl PI and incubate or 15 min at room temperature (25°C) in the dark

Publication protocol

Apoptosis induced by ZOL was quantified using the Annexin V-FITC Apoptosis Detection kit I (BD Biosciences, Franklin Lakes, NJ, USA). MG-63 cells were treated with 50 μM of ZOL for 24, 48 and 72 h and then washed twice with cold PBS and re-suspended in 1× binding buffer at a concentration of 1×106 cells/ml. The solution (100 μl, 1×105 cells) was transfered to a 5-ml culture tube and 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) were added. The cells were gently vortexed and incubated for 15 min at room temperature (25°C) in the dark. 1× binding buffer (400 μl) was added to each tube. Samples were analyzed by flow cytometry within 1 h. In the dual parameter fluorescent dot plots, the cells in early and late apoptosis were counted. Total cell apoptosis was defined as the sum of cells in early and late apoptosis. Experiments were performed in triplicate.

Confluent MG-63 cells (treated with increasing concentrations of ZOL for 24, 48 and 72 h) were removed from culture dishes by trypsinization and fixed with ice-cold 70% ethanol at −20°C overnight. Cells were washed with PBS, treated with DNase A (200 g/l, Sigma, St. Louis, MO, USA) and stained with PI (50 g/l, Sigma) at room temperature for 30 min in the dark. Cell cycle distribution was determined by the FACSCalibur flow cytometer (BD Biosciences).

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