This is a cached page for the URL (http://cancer.ucsd.edu/flow/Protocols/DNA&NuclearAg.asp). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Nuclear Antigens

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):

  1. If adherent, trypsinize and wash once with PBS (without Ca++ and M++, with 2% Bovine Serum Albumin)
  2. Pellet cells and resuspend in ice-cold Freezing Buffer to a minimum concentration of 105 cells in 50 ml and store at –80°C
  3. 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
  4. Add 25 ml FITC-conjugated antibody and mix for 30 minutes
  5. 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):

  1. 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.
  2. 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:
  1. 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
  2. Landberg, G., Roos, G. Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells. Cytometry, 1992, 13(3):230-40.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. Jacob MC; Favre M; Bensa JC. Membrane cell permeabilization with saponin and multiparametric analysis by flow cytometry. Cytometry, 1991, 12(6):550-8.


 <---Back to Flow Cyto Home

<--- Back to Protocols

Dennis J. Young, UCSD Cancer Center Flow Cytometry Core Facility


Home  |  Events  |  Friends & Supporters  |  Faculty & Staff site |  Search

This site is a service of the Moores UCSD Cancer Center.  Comments or questions?  Please contact our webmaster.
Help using this site.