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Nuclear Antigens and DNA
These washless procedures require
proper titration of antibodies!
Procedure A ( frozen cells):
- If adherent, trypsinize and wash once with PBS
(without Ca++ and M++, with 2% Bovine Serum Albumin)
- Pellet cells and
resuspend in ice-cold Freezing Buffer to a minimum concentration of 105
cells in 50 ml and store at –80°C
- While thawed cells
are slowly agitated in an ice bath mounted on a mixer, add 200 ml
Lysis-DNA Staining Solution and mix for 15 minutes
- Add 25 ml
FITC-conjugated antibody and mix for 30 minutes
- Analyze
Reagents:
Freezing Buffer:
250 mM sucrose
40 mM sodium citrate
5% v/v DMSO
pH 7.6
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Lysis-DNA Staining Solution:
0.5% Nonidet P40
20 mg/ml
propidium iodide (PI)
0.2 mg/ml RNAse
0.5 nM EDTA
pH 7.2 |
Procedure B (fresh cells):
- Suspend 2 x 105 fresh cells in PBS and mix with 100 ml
Lysing Solution supplemented with appropriate dilution of antibody and
incubate 30 min at room temperature.
- Add 100 ml
of an appropriate dilution of the secondary FITC-conjugated antibody containing
PI (final concentration 10 mg/ml) and RNAse
(final concentration 100 mg/ml) and incubate
30 minutes RT.
Reagent:
Lysing Solution:
1% Bovine Serum Albumin (BSA)
0.5% Triton
X-100
0.2 mg/ml EDTA
in PBS
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References:
- Larsen, J.K., “Washless” Procedures for Nuclear Antigen Detection. In:
Methods in Cell Biology: Flow Cytometry, Vol. 41, Chapter 24, page 377-388.
Academic Press, Inc. San Diego. 1994
- Landberg, G., Roos, G. Flow cytometric analysis of proliferation associated
nuclear antigens using washless staining of unfixed cells. Cytometry,
1992, 13(3):230-40.
- Vindelov, L.L., Christensen, I.J., Keiding, N., Spang-Thomsen, M.,
Nissen, N.I., Long-term storage of samples for flow cytometric DNA analysis.
Cytometry 3 (1983), 317-322.
- Baisch, H., Gerdes, J. Simultaneous staining of exponentially growing
versus plateau phase cells with the proliferation-associated antibody
Ki-67 and propidium iodide: analysis by flow cytometry. Cell and Tissue
Kinetics, 1987 Jul, 20(4):387-91.
- Palutke M; KuKuruga D; Tabaczka P. A flow cytometric method for measuring
lymphocyte proliferation directly from tissue culture plates using Ki-67
and propidium iodide. Journal of Immunological Methods, 1987 Dec 4, 105(1):97-105.
- Sasaki K; Murakami T; Kawasaki M; Takahashi M. The cell cycle associated
change of the Ki-67 reactive nuclear antigen expression. Journal
of Cellular Physiology, 1987 Dec, 133(3):579-84.
- Drach J; Gattringer C; Glassl H; Drach D; Huber H. The biological
and clinical significance of the KI-67 growth fraction in multiple myeloma.
Hematological Oncology, 1992 Mar-Apr, 10(2):125-34.
- Jacob MC; Favre M; Bensa JC. Membrane cell permeabilization with saponin
and multiparametric analysis by flow cytometry. Cytometry, 1991, 12(6):550-8.
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Dennis J. Young, UCSD
Cancer Center Flow Cytometry Core Facility
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