Staining Protocol

1. Resuspend the positive (brown cap) and negative (clear cap) control cells by swirling the vials. Remove 1.0 ml aliquots of the control cell suspensions (~1 x 10 6 cells/ml) and place in 12 x 75 mm centrifuge tubes. Centrifuge the control cell suspensions for 5 min at 300 x g and remove the 70% (v/v) ethanol by aspiration, being careful to not disturb the cell pellet.
2. Resuspend each tube of control cells with 1.0 ml of Wash Buffer (blue cap) for each tube. Centrifuge as before and remove the supernatant by aspiration.
3. Repeat the Wash Buffer treatment (step 2).
4. Resuspend each tube of the control cell pellets in 50 µl of the Staining Solution (prepared as described below).

5. Incubate the cells in the Staining Solution for 60 min at 37°C. The reaction can also be carried out at RT overnight for the control cells. For test samples, the 60 min incubation time at 37°C may need to be adjusted to longer periods of time.
6. At the end of the incubation time, add 1.0 ml of Rinse Buffer (red cap) to each tube and centrifuge each tube at 300 x g for 5 min. Remove the supernatant by aspiration.
7. Repeat the cell rinsing with 1.0 ml of the Rinse Buffer, centrifuge and remove the supernatant by aspiration.
   Note: PI/Rnase treatment is not necessary if cell cycle is not being analyzed (Proceed to step 11).
8. Resuspend the cell pellet in 1.0 ml of the PI/RNase A solution (amber bottle).
   Note: If the cell density is low, decrease the amount of PI/RNase A solution to 0.3 ml.
9. Incubate the cells in the dark for 30 min at RT.
10. Analyze the cells in PI/RNase A solution by flow cytometry.
11. Analyze the cells by flow cytometry within 3 hr of staining. Cells may begin to deteriorate if left overnight before analysis.