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

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. Cytometry 43:12–15, 2001. © 2001 Wiley‐Liss, Inc.