Preparation and bivariate analysis of suspensions of human chromosomes

G. J. van den Engh, B. J. Trask, J. W. Gray, R. G. Langlois, L. ‐C Yu

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)92-100
Number of pages9
JournalCytometry
Volume6
Issue number2
DOIs
StatePublished - Mar 1985

Keywords

  • Hoechst 33258
  • Human chromosomes
  • amniocytes
  • chromomycin A3
  • flow karyotypes
  • peripheral blood lymphocytes

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biophysics
  • Hematology
  • Endocrinology
  • Cell Biology

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  • Cite this

    van den Engh, G. J., Trask, B. J., Gray, J. W., Langlois, R. G., & Yu, L. C. (1985). Preparation and bivariate analysis of suspensions of human chromosomes. Cytometry, 6(2), 92-100. https://doi.org/10.1002/cyto.990060203