Suggest Fixation Protocol

Results may vary according to cell origin and growth conditions.

1. Suspend the cells in 1% (w/v) paraformaldehyde in PBS (pH 7.4) at a concentration of 1-2 x 10 6 cells/ml.
2. Place the cell suspension on ice for 30-60 minutes.
3. Centrifuge cells for 5 min at 300 x g and discard the supernatant.
4. Wash the cells in 5 ml of PBS, then pellet the cells by centrifugation. Repeat the wash and centrifugation.
5. Resuspend the cell pellet in the residual PBS in the tube by gently vortexing the tube.
6. Adjust the cell concentration to 1-2 x 10 6 cells/ml in 70% (v/v) ice cold ethanol. Let cells stand for a minimum of 30 min on ice or in the freezer. See note below.
7. Store cells in 70% (v/v) ethanol at -20°C until use. Cells can be stored at -20°C several days before use.

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.

The following protocol describes the method for measuring apoptosis in the positive and negative control cells that are provided in the APO-BRDU™ Kit. The same procedure should be employed for measuring apoptosis in the cell specimens provided by the researcher.

Staining Protocol

1. Resuspend the positive (brown cap) and negative (white cap) control cells by swirling the vials. Remove 1 ml aliquots of the control cell suspensions (approximately 1 x 10 6 cells/ml) and place in 12 x 75 mm flow cytometry 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 DNA Labeling Solution (prepared as described below).
5. Incubate the cells in the DNA labeling Solution for 60 min at 37°C in a temperature controlled bath. Shake cells every 15 min to resuspend.

*The appropriate volume of DNA Labeling Solution to prepare for a variable number of assays is based upon multiples of the component volumes needed for 1 assay. Mix only enough DNA Labeling Solution is active for approximately 24 hr at 4° C.

NOTE: The DNA Labeling Reaction can also be carried out at RT overnight for the control cells. For samples other than the control cells provided in the kit, incubation times at 37°C may need to be adjusted to longer or shorter periods depending on the requirements of the cells.

1. 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.
2. Repeat the cell rinsing (as in step 6) with 1.0 ml of the Rinse Buffer, centrifuge and remove the supernatant by aspiration.
3. Resuspend the cell pellet in 0.1 ml of the Antibody Solution (prepared as described below).

1. Incubate the cells with the FITC labeled anti-BrdU Antibody Solution in the dark for 30 min at RT.
   Hint: Wrap tubes with aluminum foil to protect from light.
   Note: PI/Rnase treatment is not necessary if cell cycle is not being analyzed (Proceed to step 13).
2. Add 0.5 ml of the PI/RNase A Solution (amber bottle) to the tube containing the 0.1 ml Antibody Staining Solution.
   Note: If the cell density is low, decrease the amount of PI/RNase A solution to 0.3 ml.
3. Incubate the cells in the dark for 30 min at RT.
4. Analyze the cells in PI/RNase A Solution by flow cytometry.
5. Analyze cells within 3 hr of staining to obtain optimal results. Cells may begin to deteriorate if left overnight before analysis.