|Application Name||Yes||No||Not Determined||Suggested Dilution|
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
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 by pipetting up and down.
3) Resuspend cells in 200ul pre-diluted binding buffer, adjusting to a cell density of 2-5 x 105 cells/ml.
4) Add 5ul Annexin V:FITC to 195ul of the cell suspension prepared in step 3.
5) Mix and incubate for 10 minutes in the dark, at room temperature.
6) Wash cells in 200ul of pre-diluted binding buffer.
7) Resuspend cells in 190ul pre-diluted binding buffer.
8) Add 10ul of the Propidium Iodide solution.
9) 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.