Instructions For Use
1) Dilute the binding buffer 1:4 in distilled water (50ml binding buffer + 150ml distilled water).
2) Wash cells in PBS by gentle shaking or pipetting up and down.
3) Resuspend cells in 200ul pre-diluted binding buffer, adjusting to a cell density of 2-5 x 105
4) Add 5ul Annexin V:Biotin to 195ul of the cell suspension prepared in step 3.
5) Mix and incubate for 15 minutes at room temperature.
6) Wash cells twice in 190ul of pre-diluted binding buffer.
7) Resuspend cells in 190ul pre-diluted binding buffer.
8) Add streptavidin:FITC conjugate.
9) Mix and incubate for 30 minutes in the dark, at room temperature.
10) Wash cells in 200ul pre-diluted binding buffer.
11) Resuspend cells in 190ul pre-diluted binding buffer.
12) Add 10ul of the Propidium Iodide solution.
13) Analyse by flow cytometry.
The flow cytometer is preferably set such that the Mean Fluorescence Intensity of the Annexin V negative population is between 1 and 10. Optimal parameter settings can be found using a positive control. For a positive control, incubate the cells with 3% formaldehyde in buffer during 30 minutes on ice. Wash away the formaldehyde and suspend the cells in cold binding buffer at 2-5 x 105
cells/ml. Proceed with step 2 as described above.