For Cat. No. 556381, see Technical Data Sheet for methods.
- Suspend the cells in 1% (w/v) paraformaldehyde in PBS (pH 7.4) at a concentration of 1-2 x 10 6 cells/ml.
- Place the cell suspension on ice for 30-60 minutes.
- Centrifuge cells for 5 min at 300 x g and discard the supernatant.
- Wash the cells in 5 ml of PBS, then pellet the cells by centrifugation. Repeat the wash and centrifugation.
- Resuspend the cell pellet in the residual PBS in the tube by gently vortexing the tube.
- Adjust the cell concentration to 1-2 x 10 6 cells/ml in 70% (v/v) ice cold ethanol. Let cells stand for a minumum of 30 minutes on ice or in the freezer. See note below.
- Let cells stand for a minimum of 30 min on ice or in the freezer.
Note: In some biological systems storage of the cells at -20°C in 70% (v/v) ethanol for at least 12–18 hr prior to staining for apoptosis detection yields the best results. Cells can be stored at -20°C for several months before use.
- 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.
- 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.
- Repeat the Wash Buffer treatment (step 2).
- Resuspend each tube of the control cell pellets in 50 µl of the Staining Solution (prepared as described below).
- 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.
- 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.
- 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).
- 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.
- Incubate the cells in the dark for 30 min at RT.
- Analyze the cells in PI/RNase A solution by flow cytometry.
- Analyze the cells by flow cytometry within 3 hr of staining. Cells may begin to deteriorate if left overnight before analysis.