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DNA/PI Staining for Cell Cycle

Materials:

  • 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

Protocol:

  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.

Reagents:

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.