Cell Fixation Procedure for APO-BRDUTM ASSAY

NOTE: Cell fixation using paraformaldehyde is a required step in the APO-BRDUTM assay. The following cell fixation procedure is a suggested method. Variables such as cell origin and growth conditions can affect the results. The fixation conditions provided below should be considered as guidelines. Additional experimentation may be required to obtain results comparable to the control cells provide with this kit. The positive and negative control cells provided in the APO-BRDUTM KIT are already fixed.
  1. Suspend 1-2 x 106 cells in 0.5 ml of 10 mM sodium phosphate pH 7.2, 150 mM sodium chloride (PBS).

  2. Add the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes.

  3. Centrifuge cells for 5 minutes 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 cells in 0.5 ml of PBS.

  6. Add cells to 5 ml of ice-cold 70% (v/v) ethanol. Let cells stand for a minimum of 30 min. in ice or the freezer. See note below.

  7. Store cells in 70% (v/v) ethanol at -20C until use. Cells can be stored at -20°C several days before use.
Note: In some biological systems storage of the cells at -20C in 70% (v/v) ethanol for at least 12-18 hours prior to staining for apoptosis detection yields the best results.