| Flow cytometry main page Protocols main page | <---Back to Flow Cyto Home <--- Back to Protocols 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 | 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 | 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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<--- Back to Protocols Dennis J. Young, UCSD Cancer Center Flow Cytometry Core Facility |
