Supported by the Danish Medical Research Council, the Danish Cancer Society, the Simon Spies Foundation, and the Mogens and Jenny Vissing Foundation.
Article first published online: 8 MAR 2005
Copyright © 1991 Wiley-Liss, Inc.
Volume 12, Issue 5, pages 429–437, 1991
How to Cite
Larsen, J. K., Christensen, I. J., Christiansen, J. and Mortensen, B. T. (1991), Washless double staining of unfixed nuclei for flow cytometric analysis of DNA and a nuclear antigen (Ki-67 or bromodeoxyuridine). Cytometry, 12: 429–437. doi: 10.1002/cyto.990120508
This method was first presented at EACR-IX, Helsinki, May 1987, and SAC-XII, Cambridge, August 1987.
- Issue published online: 21 JUN 2005
- Article first published online: 8 MAR 2005
- Manuscript Accepted: 11 MAR 1991
- Manuscript Received: 18 NOV 1988
- Ki-67 antibody;
- anti-bromodeoxyuridine antibody;
- DNA synthesis;
- cell cycle;
- cell proliferation;
- lymphocyte activation;
- HL-60 cells
Washless methods for double staining of nuclear antigen and DNA in unfixed nuclei were compared with established methods for staining of fixed cells. The methods were tested on phytohemagglutinin (PHA)-stimulated normal human blood lymphocytes for the double staining of (1) Ki-67 antigen and DNA and (2) bromodeoxyuridine (BrdUrd) and DNA, in continuously BrdUrd-labeled cells. With respect to the discrimination between antigen-positive and -negative subpopulations, there was no statistically significant differences between the results from direct (Ki-67) or indirect, (Ki-67 or BrdUrd) washless staining of unfixed nuclei and the results from staining of fixed cells. Washless staining of unfixed nuclei was found to be rapid and simple and resulted in greater precision of the DNA analysis and in less aggregation and loss of cells.