**These protocols are for staining the DNA of all cells in a sample for use in DNA cell cycle analysis. They are not recommended for apoptosis analysis, except as a preliminary check for DNA degradation (subG0). They are also not appropriate to check viability, since the samples are fixed in EtOH prior to staining with PI.**
Step 1: Harvest and count cells.
Step 2: Centrifuge and remove supernatant. Vigorously vortex the pellet for 10 seconds and continue to vortex the cells while slowly adding 1 ml of ice cold 70% ethanol drop by drop to the pellet. Fix overnight at 4 o C.
Step 3: Prepare PI staining solution (prepare fresh for each staining assay).
-8.5 ml staining buffer (0.1% BSA in PBS)
-1 ml Rnase (stock at 1% – 1:10 = 0.1%)
-0.5 ml PI stock solution (stock at 1 mg/ml)
Step 4: Briefly vortex tube with sample. Centrifuge 10 minutes at high speed (3000 rpm) and remove tube. Pour off ethanol. Leave less than or equal to 0.2 ml ethanol in tube.
Step 5: Gently vortex tube to suspend cells in residual ethanol. Add 0.5 to 1.0 ml PI staining solution to each tube and vortex. Let sample incubate for greater than or equal to 30 minutes at room temperature in the dark.
Step 6: Transfer to 5 ml collection tubes (12 x 75 mm), if necessary, and analyze on the FACSCalibur in the UAMS Flow Cytometry Core Facility within 24 hours.
Protocol 2 Fixation
Transfer the medium with floating cells (4ml from 60mm dish) into 15 ml centrifuge tube.
Wash the attached cells with 2 ml PBS, and add the PBS to the 15ml tube containing the medium and floating cells.
Add 0.7 ml Trypsin to the plate to detach the cells. Stop the digestion by adding the 6 ml of medium with floating cells from the 15ml tube. Transfer the 6ml medium, now containing all the cells, to the 15 ml tube.
Spin down all the cells at 3000 rpm for 5 min.
Discard the medium and wash the cells with PBS two times (spinning down all the cells at 3000 rpm for 5 min after each wash).
Resuspend the cells in 0.7 ml PBS, vigorously vortex the pellet for 10 seconds, and add 10 ml 75% ethanol – vortex to mix briefly.
Fix cells at 4 o C O/N or until ready to stain.
Briefly vortex then spin fixed cells down at 3000 rpm for 5 min. Pour off ethanol. Leave less than 0.2 ml ethanol in tube.
Resuspend cells in 0.5 – 1 ml (depending cell number, minimal 3×105 cell in 0.5 ml) 1 X PI (Propidium Iodide) in PBS working Solution (50 m g/ml) by diluting 20 X stock (1 mg/ml). Add 15 m l/ml RNaseA (7 mg/ml). Transfer to 12x75mm 5 ml tube (if see clumps, pass into the 5 ml tube with a cell-strainer cap 2235).
Incubate at 37 o C for 30 min
Analyze on FACSCalibur within 24 hours
Propidium Iodide (Cat. P 4170) and RnaseA ( Cat. R 6513 ) are from Sigma Chemicals.
P.I. Stock Solution (1 mg/ml in water)
RNaseA solution (7 mg/ml in 10 mM Tris.HCl pH 7.5, 15 mM NaCl)
120 ml working Staining Solution
6 ml 20 X P.I. Stock (1mg/ml) (store in 4 º C)
1.8 ml RNase A (7 mg/ml) (store at -20 ºC)
113 ml PBS