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DNA Content of Adherent Cells

Reagents

Cryopreservative:

  • 250 mM sucrose
  • 5% dimethylsulfoxide (DMSO) v/v
  • 40 mM trisodium citrate
  • pH 7.6

Stock Solution:

  • 3.5 mM trisodium citrate
  • 0.1% v/v Nonidet P-40
  • 0.5 mM TRIS
  • 1.5 mM spermine tetrahydrochloride
  • pH 7.6

RNAse Solution:

  • 500 µg/ml trypsin inhibitor
  • 10 µg/ml RNAse

Propidium Iodide (PI) Solution:

  • 42 µg/ml PI
  • 1.16 mg/ml spermine tetrahydrochloride

Procedure

  1. Remove media from culture dish and rinse cells with 1.0 ml of PBS.
  2. Add 500 µl of Cryopreservative.
  3. Store dishes at -80°C until ready to stain.
  4. Thaw at room temperature and remove Cryopreservative.
  5. Add 900 µl Trypsin Solution (30µg/ml trypsin in Stock Solution).
  6. Mix gently and keep at room temperature for 10 min.
  7. Add 750 µl RNAse Solution.
  8. Mix gently and keep at room temperature for 10 min.
  9. Add 750 µl PI Solution.
  10. Filter solution through Falcon® Cell Strainer Cap (Cat. No. [35]2235) and keep on ice protected from light for at least 30 min & up to 3 hours.

Reference

Tennenbaum T, Giloh H, Fusenig NE, Kapitulnik J: A rapid procedure for the flow cytometric DNA analysis in cultures of normal and transformed epidermal cells. Journal of Investigtive Dermatology, 90:857-860, 1988.