Principles:

PI dyes the DNA which can indicates the cell cycle phases (G1, G2/M), Using Sub-G1 phase as an indicator of apoptosis.

Required reagents

1) RNase A: 10 mg/ml in 10mM Tris-HCl, pH7.5

2) PI: 500 μg/ml in 38 mM sodium citrate, pH7.0 or in PBS

3) Ethanol 75%

4) PBS+ 1% BSA (0.22 μm filtration)

Procedure

1. Harvest Cells in 15 ml tube, make sure to make single cell suspension (check the cells under microscope to see the cells are single);

2. Centrifuge @1000 rpm, 5 min, 4 ℃, and remove supernatant;

3. Wash twice in PBS, and count the cell number (1×10⁶ cells);

4. Resuspend the pellet in 500 μl PBS (single cell suspension);

5. Add cell suspension into 5 ml COLD (-20℃) 75% ethanol (very slowly , drop by drop ). Fix at 4 ℃ overnight (Cells can remain in fixative up to 3 weeks before staining);

6. Centrifuge at 1000 rpm, 10 min, 4 ℃, remove ethanol (very gentle, avoid disturbing the cell pellet， MAY not see pellet after ethanol fixation);

7. Wash twice with 5 ml PBS+1% BSA;

8. Resuspend cells in 400 μl PBS+1% BSA (could reduce the volume to 200 μl);

9. Add 50 μl 500 μg/ml PI (could reduce accordingly if the cell number is low);

10. Add 50 μl boiled 10 mg/ml RNase A (may be omitted if number of cells is low);

11. Incubate at 37 ℃ for 30 min (samples can be stored and protected from light at 4℃);

12. FACS, using FL2 histogram (620 nm wavelength).