Fixation-associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs.
We have examined, by flow cytometry, the apparent DNA content of frog blood cells that had been fixed with either 50% ethanol, 70% ethanol, or 66% methanol, before being stained with either mithramycin, propidium iodide, or Hoechst 33258. After 50% ethanol fixation, regardless of the dye used, the DNA content of the hemopoietic cells appeared unimodal, but after either 70% ethanol or 66% methanol fixation, it appeared bimodal. Cell sorting revealed that the lower and upper modes are represented by erythrocytes (RBCs) and leukocytes (WBCs), respectively. In amphibians, the chromatin of metabolically inactive RBCs is highly condensed relative to the chromatin of metabolically active WBCs. The bimodal distribution of DNA contents seen with 66% methanol and 70% ethanol, but not 50% ethanol, seems to reflect this disparity in the degree of chromatin condensation existing between the RBCs and WBCs. This, in turn, implies that the accessibility of fluorescent DNA dyes to the chromatin of fixed frog hemopoietic cells, especially of RBCs, can be affected by the concentration of alcohol used for their fixation.
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, New York 14642.
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Holtfreter
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Cohen
D000818:Animals; D006690:Bisbenzimidazole; D002678:Chimera; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000097:blood*; D000431:Ethanol; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006413:Hematopoietic System / Q000166:cytology; D000432:Methanol; D008926:Plicamycin; D011003:Ploidies; D011419:Propidium; D011894:Rana pipiens / Q000097:blood*; D015203:Reproducibility of Results; D014982:Xenopus laevis / Q000097:blood*
D002843:Chromatin; D005404:Fixatives; D005456:Fluorescent Dyes; D011419:Propidium; D000431:Ethanol; D004247:DNA; D006690:Bisbenzimidazole; D008926:Plicamycin; D000432:Methanol