NASA/American Cancer Society high-resolution flow cytometry project - II. Effect of pH and DAPI concentration on dual parametric analysis of DNA/DAPI fluorescence and electronic nuclear volume

Jinghai Wen, Awtar Krishan, Richard A. Thomas

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

Background: In the present paper, we describe the effect of 4', 6-diamidino-2-phenylindole (DAPI) dihydrochloride concentration and pH on the resolution of DNA distribution histograms generated by dual-parametric simultaneous analysis of DNA content and electronic nuclear volume (ENV). Methods: Nuclei from tissue culture cell lines and frozen human solid tumors were isolated in nuclear isolation media containing different concentrations of DAPI, at various pH levels, and analyzed on a NASA/American Cancer Society (ACS) flow cytometer. Samples stained with propidium iodide/hypotonic citrate and analyzed in a Coulter XL flow cytometer were used for comparison. Results: Nuclei stained with DAPI concentration of 1-3 μg/ml, pH 6.0, gave the best resolution for the detection of the near-diploid and near-tetraploid populations. Simultaneous use of ENV and DAPI/DNA fluorescence under these conditions identified subpopulations that otherwise could not be detected by DNA analysis alone. Conclusions: Staining at 1-3 μg/ml DAPI, pH 6.0, was optimal for the detection of aneuploid populations, especially the near-diploid and/or near-tetraploid populations in human tumors.

Original languageEnglish (US)
Pages (from-to)12-15
Number of pages4
JournalCytometry
Volume43
Issue number1
DOIs
StatePublished - Jan 1 2001

Keywords

  • Aneuploidy
  • DAPI
  • DNA index
  • Electronic nuclear volume
  • Flow cytometry
  • Fluorochromes
  • pH

ASJC Scopus subject areas

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

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