Fixation‐Associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs

Abstract

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.Key terms: DNA fluorescent dye accessibility, fixation artifacts, frog hemopoietic cells, erythrocyte chromatin compaction, ploidy We and others have been using diploidhiploid chimeras to investigate the developmental origin of hemopoietic cell in the frogs, Rana pipiens and Xenopus laeuis. Chimerism was established prior to larval life by fusing defined regions of diploid (2N) and triploid (3N) tailbud stage embryos (3,11,22). In principle, the ploidy of blood cells in chimeric tadpoles or adults should indicate the embryonic site(s) of that cell type. During the course of some control studies that involved different fixation protocols, we noted that the amount of DNA in RBCs and WBCs of normal (i.e., non-chimeric) 2N or 3N perimetamorphic tadpoles, froglets, and mature frogs appeared to differ considerably as if the RBCs and WBCs from a single animal were of a different ploidy. These observations compelled us to investigate the possibility of fixation artifacts, and to establish a reproducible fixation protocol for flow cytometry that allows fluorescent DNA dyes to bind stoichiometrically to the chromatin of all hemopoietic cells (i.e., independent of their developmental stage or degree of chromatin compaction) such that the dye bound to the chromatin accurately reflects the cells' ploidy. MATERIALS AND METHODSMaterials Non-chimeric diploid adult frogs, Rana pipiens (J.M. Hazen and Co., Alburg, VT) and Xenopus laeuis (South African Snake Farm, Capetown, South Africa), were used. In addition, a few non-chimeric 3N adult Xenopus laeuis (9,24) were also used. MethodsCell suspension medium (CSM). Hemopoietic cells were suspended in Leibovitz-15 medium (L-15) (Gibco, Grand Island, NY) that was diluted to the osmolarity of adult frog serum (220 mOsm) and supplemented with 1.25 x lop5 M HEPES buffer, 100 U/ml penicillin, 100 kg/ml streptomycin, and 2% fetal bovine 'Address reprint requests to Dr.

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