Summary
Liver cells were isolated by a collagenase perfusion technique. A fraction of the cell suspension containing mostly parenchymal cells was obtained by slow speed centrifugation. Separated, intact cells were stained with mitramycin for flow cytometry. A brief pre-treatment with toluene was found necessary for rapid and uniform staining. Isolated nuclei were prepared by a combination of detergent and pepsin treatments and stained with ethidium bromide.
Flow cytometric histograms of isolated liver cells from adult animals gave peaks at the 2c, 4c, 8c and 16c levels. Owing to the presence of binucleate cells, the preparations of intact cells showed a higher number of cells in the higher ploidy classes than preparations of isolated nuclei. The number of diploid pulses corresponded to the admixture of non-parenchymal cells, strongly suggesting that the adult mouse liver contains very few diploid parenchymal cells or none at all.
The ethidium bromide-stained nuclei were sorted on a cell sorter according to dye binding, further examined by light microscopy and subjected to biochemical DNA analyses. Light microscopy of isolated nuclei revealed that most of the nuclei from so-called binucleate cells were held together by pepsin-resistant filaments. Some of the parenchymal cell nuclei were lobulated. On the basis of these observations, the real ploidy distribution was discussed and compared with previous results obtained by Feulgen microspectrometry. The stoichiometry of dye binding was confirmed for the diploid (non-parenchymal) nuclei and the tetraploid (parenchymal) nuclei by performing biochemical DNA measurements on sorted material.
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Research fellow supported by the Norwegian Council for Science and the Humanities
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Digernes, V., Bolund, L. The ploidy classes of adult mouse liver cells. Virchows Archiv B Cell Pathol 32, 1–10 (1980). https://doi.org/10.1007/BF02889008
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DOI: https://doi.org/10.1007/BF02889008