Cell Cycle Analysis
Prior to sending samples to MDS for FACS analysis, prepare cells as described below:
Isolate the Cells
Grow cells to less than 2 x 107 cells/mL
- For adherent cells, remove with PBS/EDTA and/or trypsin solution.
- For cells in suspension, harvest by centrifugation.
- Centrifuge cells at 1200 rpm at 4°C for 5 minutes, decant supernatant and gently re-suspend the cells in PBS.
- Count the cells by hemocytometer.
- Wash cells one time by putting 1 x 105 cells per tube, adding 1 mL of PBS and centrifuging at 1200 rpm at 4°C. Re-suspend pelleted cells in 0.3 mL of PBS buffer.
- To fix the cells, gently add 0.7 mL cold ethanol (70%) drop wise to tube containing 0.3 mL of cell suspension in PBS while vortexing gently.
- Leave on ice for 1 hour (or up to a few days at 4°C).
- Centrifuge cells as above, wash 1 time with cold PBS and re-centrifuge.
- Re-suspend cell pellet in 0.25 mL of PBS and add 5 µL of 10 mg/mL Proteinase K (the final concentration being 0.2-0.5 mg/mL).
- Incubate at 37°C for 1 hour.
- Add 10 µL of 1 mg/mL Presidium iodide (the final concentration being 10 µg/mL) solution.
- Incubate for 2 to 2.5 hr at 50oC
- Add 1 mL of 16 µg/mL propidium iodide in 50 mM sodium citrate (made from 100x propidium iodide stock stored at -20oC). 1 M Sodium Citrate buffer stock - Make 1 M NaCitrate. Carefully adjust the 50 mM dilution with a few grains of citric acid crystals to reach pH 7.4. Store at room temperature.
- Cells may be sonicated or vortexed again if required.
- Let stand at room temperature for 30 min.
- Keep away from light. You can store for 1 week at 4oC
- Send to MDS for FACS analysis