Cell Cycle Analysis

Isolate 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 1X105 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.


Fixing the Cells

. 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, 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 PI (Presidium iodide, the final concentration being 10µg/ml) solution.
. Incubate for 2 to 2.5 h at 50 degrees C
. Add 1 ml of 16 µg/ml propidium iodide in 50 mM NaCitrate (made from 100x propidium iodide stock stored @ -20 degrees C). 1 M 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 temp
. Cells may be sonicated or vortex again if required.
. Let stand at room temperature for 30 min.
. Keep away from light. You can store for 1 week at 4 degrees C
. Send to MDS for FACS analysis