DNA distribution analysis of type II pneumocytes by laser flow cytometry: technical considerations

Optimal conditions were established for determination of cell cycle phase fractions of freshly isolated or cultured adult rat type II pneumocytes (T2P). Propidium iodide staining of ethanol-fixed cells treated with ribonuclease (RNase) consistently yielded histograms with low coefficients of variation. Contaminating cells and cell clumps were eliminated during data acquisition through electronic gating based on anti-vimentin immunofluorescence and peak red fluorescence, respectively. Failure to delete contaminants, clumps or RNA resulted in overestimation of S or G2/M phase fractions by as much as 20-fold. When T2P were cultured on plastic at an initial density of 2.5 x 10(5)/cm2, the S phase fraction did not change over a culture interval in which thymidine incorporation rates increased almost 10-fold. In contrast, a significant increase in the G2/M phase fraction by day 2 of culture occurred with no significant increase in cell number. These results support the hypothesis that adult rat T2P, when subjected to customary conditions of primary culture, undergo cell cycle block in G2/M phases. The data also indicate that under these in vitro conditions, net thymidine incorporation by T2P may vary independently of the S phase fraction. The methods described in this report address basic considerations crucial to future applications of flow cytokinetics to the study of T2P proliferation and differentiation.