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

Background In the present paper, we describe the effect of 4', 6-diamidino- 2-phenylindole (DAPT) dihydrochloride concentration and pH on the resolutio n 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 tumor s were isolated in nuclear isolation media containing different concentrati ons of DAPI, at various pH levels, and analyzed on a NASA/American Cancer S ociety (ACS) now cytometer. Samples stained with propidium iodide/hypotonic citrate and analyzed in a Coulter XL now cytometer were used for compariso n. Results: Nuclei stained with DAPT concentration of 1-3 mug/ml, pH 6.0, gave the best resolution for the detection of the near-diploid and near-tetrapl oid populations. Simultaneous use of ENV and DAPI/DNA fluorescence under th ese conditions identified subpopulations that otherwise could not be detect ed by DNA analysis alone. Conclusions: Staining at 1-3 mug/ml DAPI, pH 6.0, was optimal for the detec tion of aneuploid populations, especially the near-diploid and/or near-tetr aploid populations in human tumors. Cytometry 43:12-15, 2001. (C) 2001 Wile y-Liss, Inc.