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:
- Remove
media from culture dish and rinse cells with 1.0 ml of PBS.
- Add 500 µl
of Cryopreservative.
- Store
dishes at -80°C until ready to stain.
- Thaw at
room temperature and remove Cryopreservative.
- Add 900 µl
Trypsin Solution (30µg/ml
trypsin in Stock Solution).
- Mix gently
and keep at room temperature for 10 min.
- Add 750 µl
RNAse Solution.
- Mix gently
and keep at room temperature for 10 min.
- Add 750 µl
PI Solution.
- 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.
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Dennis J.
Young, UCSD Cancer Center Flow Cytometry Core Facility
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