DNA Cell cycle analysis using Propidium Iodide

Developed by: Justin Meyers

Sample: Cell line or primary cells

Materials

• Propidium iodide reagent (Sigma P-4170) 10mg make up 1mg/ml solution in DI water and store at 4C
• Rnase A, (Sigma R-6513), 10mg, store at -20C

Procedure

Isolate cells

1. Harvest cells, want good single cell suspension
2. Count cells
3. Dispense aliquots of 1 million cells into labeled tubes
4. Add 5 ml PBS and centrifuge for 5 min at 400 x g.
5. Re-suspend cell pellet in 300 ul PBS with gentle vortex

Fix cells:

1. Add 700 ul ice cold ethanol (200 proof), slowly, while continuously vortexing.
2. Incubate at 4C for 30 min to overnight
3. Pellet cells at 2000 rpm for 5 min
4. Re-suspend (vortex gently) cells in 250 ul of PBS
5. Add 5ul of 10mg/ml Rnase A (final concentration being 0.2-0.5 mg/ml)
6. Incubate at 37C for one hour
7. Add 10ul of the 1mg/ml PI solution (final concentration is 10ug/ml)
8. Keep in the dark for at least one hour until analysis
9. Perform flow cytometry analysis on flow