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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Cellular complexes, analogous by virtue of their external appearance, size, and number of seemingly internalized thymocytes to thymic nurse cells (TNCs) of endothermic vertebrates, were seen in short-term cultures (6-8 days) of mechanically and enzymatically dissociated thymuses of leopard frog tadpoles. Most TNC-like complexes from mechanically disrupted thymuses were covered with many thymocytes that morphologically resembled the "internalized" thymocytes. With time in culture, most complexes remained spherical and lost their externally adherent and "internalized" thymocytes. Some complexes, however, adhered to the glass substratum by means of macrophage-like cells. After one typically appearing TNC from a mechanically dissociated thymus had released its "internalized" thymocytes and spread completely over the glass substratum, it could be seen to consist actually of 9-10 stromal cells with the appearance of epithelial cells, macrophages, and dendritic cells. TNC-like structures from enzymatically dissociated thymuses had few, if any, attached thymocytes. Although these structures closely resembled murine TNCs initially, they displayed abnormal transformations within a few days of culture. Our observations led us to question the assumption that all TNCs from mechanically as well as enzymatically isolated TNCs from vertebrate thymuses are single cells. Rather, some if not all of the so-called TNC may actually be entities composed of several stromal cell types that enclose thymocytes. We suggest that this configuration seen in vitro may reflect the architecture of the compartmentalized reticular stromal cell meshwork that characterizes the intact thymus.

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