The proliferative response of rat liver parenchymal cells after partial hepatectomy A methodological study comparing flow cytometry of nuclear DNA content and in vivo and in vitro uptake of thymidine

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Abstract
DNA synthesis in rat hepatocytes, from livers regenerating after 70% hepatectomy, was assessed by flow cytometric determination of nuclear DNA content and by incorporation of [3H]thymidine. Parenchymal liver cells were isolated by collagenase perfusion and low-speed centrifugation. Nuclei from the isolated cells were prepared for flow cytometry by a treatment with detergent, pepsin and RNase and stained with ethidium bromide. Parallel samples of cells were incubated with [3H]thymidine and analyzed for rate of incorporation of radioactivity into DNA and for labeling index determination. The flow cytometric measure of the replicative response, i.e., the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [3H]thymidine. For each of 14 animals, including 2 control rats and 12 partially hepatectomized animals killed either before (at 13 h after hepatectomy), at the onset (16 and 18 h) or at the peak (24 h) of regenerating activity, a fairly good correlation was found between the different methods. Satisfactory resolution of the flow cytometric detection of S-phase cells was indicated by a sorting experiment using an Ortho (system 50-H) cell sorter, which demonstrated that after [3H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labeled, while labeling in the G1-fractions was only 2 and 7%, respectively.

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