Work performed under the auspices of the U.S. Dept. of Energy contract W-7405-ENG-48 with support from USPHS grant HD 17665 G.v.d.E. was supported by a fellowship from the Netherlands Organization for the Advancement of Pure Research (ZWO).
Preparation and bivariate analysis of suspensions of human chromosomes†
Article first published online: 8 MAR 2005
Copyright © 1985 Wiley-Liss, Inc.
Volume 6, Issue 2, pages 92–100, March 1985
How to Cite
van den Engh, G. J., Trask, B. J., Gray, J. W., Langlois, R. G. and Yu, L.-C. (1985), Preparation and bivariate analysis of suspensions of human chromosomes. Cytometry, 6: 92–100. doi: 10.1002/cyto.990060203
- Issue published online: 16 JUN 2005
- Article first published online: 8 MAR 2005
- Manuscript Accepted: 29 OCT 1984
- Manuscript Received: 14 AUG 1984
- Human chromosomes;
- flow karyotypes;
- Hoechst 33258;
- chromomycin A3;
- peripheral blood lymphocytes
Chromosomes were isolated from a variety of human cell types using a HEPES-buffered hypotonic solution (pH 8.0) containing KCl, MgSO4, dithioerythritol, and RNase. The chromosomes isolated by this procedure could be stained with a variety of fluorescent stains including propidium iodide, chromomycin A3, and Hoechst 33258. Addition of sodium citrate to the stained chromosomes was found to improve the total fluorescence resolution. High-quality bivariate Hoechst vs. chromomycin fluorescence distributions were obtained for chromosomes isolated from a human fibroblast cell strain, a human colon carcinoma cell line, and human peripheral blood lymphocyte cultures. Good flow karyotypes were also obtained from primary amniotic cell cultures. The Hoechst vs. chromomycin flow karyotypes of a given cell line, made at different times and at dye concentrations varying over fourfold ranges, show little variation in the relative peak positions of the chromosomes. The size of the DNA in chromosomes isolated using this procedure ranges from 20 to over 50 kilobases. The described isolation procedure is simple, it yields high-quality flow karyotypes, and it can be used to prepare chromosomes from clinical samples.