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DNA/PI staining for cell cycle


  • Staining medium (SM) with azide
  • 80% Ethanol
  • 2 mg/ml RNAse solution
  • 0.1 mg/ml PI solution
  • Nitex mesh (85- µm pore size)
  • FACS tubes [Falcon 2052]

Prepare in advance:

  1. RNASE solution: 2mg/ml RNAse A (Bovine Pancreas Type II, Sigma) in HBSS. Divide into 500ul aliquots and freeze.
  2. Propidium iodide solution: 0.1mg/ml PI (Sigma) in HBSS with 0.6% NP-40.
  3. HBSS Staining Medium (SM) with 2% calf serum


  1. Collect 2x106 cells by centrifugation at 400 xg for 5 minutes at 4°C. Aspirate supernatant.
  2. Spin and re-suspend pellet in 50 µl SM/CS and then add it to a conical polypropylene tube containing 1 ml of ice-cold 80% ethanol. Vortex quickly. Fix for 30 minutes to overnight at 4°C.
  3. Collect fixed cells by centrifugation at 400 xg for 5 minutes at 4°C. Aspirate supernatant. Re-suspend cell pellet in 500 µl 2 mg/ml RNase A solution. Incubate for 5 minutes at room temperature.
  4. Add 500 µl 0.1 mg/ml PI solution. Vortex to mix. Incubate for 30 minutes at room temperature, protected from light.
  5. Centrifuge at 400 xg for 5 minutes at 4°C. Re-suspend cells in 500 µl SM by gentle vortexing.
  6. Filter cell suspension through Nitex into FACS tubes.
  7. Collect data on cytometer using CELLFIT software.


Staining medium (SM) with azide

  • 1X Hank's balanced salt solution (HBSS) [InVitrogen/Gibco 14025-092]
  • 10 mM HEPES•NaOH, pH 7.2
  • 2% (v/v) calf serum
  • 10 mM NaN3
  • To 480 ml sterile 1X HBSS add 5 ml sterile 1 M HEPES•NaOH, pH 7.2, 10 ml sterile FBS and 5 ml sterile 1 M NaN3. Store at 4°C.

2 mg/ml RNAse solution

Prepare DNAse-free RNAse from bovine pancreatic RNAse, type IIA [Sigma R 5000]. Dissolve RNAse at 10 mg/ml in 10 mM sodium acetate, pH 5.2. Heat at 100°C for 15 minutes. Allow to cool to room temperature. Adjust pH to 7.4 by the addition of 1/10 volume 1 M Tris-HCl, pH 7.4. Aliquot and store at -20°C.

Alternatively, purchase RNAse A solution from Qiagen (100mg/ml stock, 17,500 units, Cat #19101). This stock can be stored at room temperature.

0.1 mg/ml PI solution

  • 0.1 mg/ml propidium iodide [Molecular Probes, P1304]
  • HBSS (no serum)
  • 0.6% (w/v) NP-40
  • Aliquot at 500 µl/aliquot and store, protected from light, at -20°C.