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:
- RNASE solution: 2mg/ml RNAse A (Bovine Pancreas Type II, Sigma) in HBSS. Divide into 500ul aliquots and freeze.
- Propidium iodide solution: 0.1mg/ml PI (Sigma) in HBSS with 0.6% NP-40.
- HBSS Staining Medium (SM) with 2% calf serum
- Collect 2x106 cells by centrifugation at 400 xg for 5 minutes at 4°C. Aspirate supernatant.
- 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.
- 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.
- Add 500 µl 0.1 mg/ml PI solution. Vortex to mix. Incubate for 30 minutes at room temperature, protected from light.
- Centrifuge at 400 xg for 5 minutes at 4°C. Re-suspend cells in 500 µl SM by gentle vortexing.
- Filter cell suspension through Nitex into FACS tubes.
- 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.