Generation of thymocyte subpopulations in organ culture: correlated analysis of Lyt-2 phenotype and cell cycle status by flow microfluorometry.

We have investigated the cell cycle kinetics and the proliferative activity of Lyt-2-defined subpopulations in organ-cultured thymic rudiments. Fetal thymuses that were removed at 13 days of gestation and cultured for 4 to 7 days were incubated with bromodeoxyuridine and were then stained with Hoechst 33342 to quantitate the duration of the cell cycle and the number of cycling cells. The total duration of the cell cycle was 30 hr. The sorting of organ culture cells according to their Lyt-2 phenotype and their forward light scatter, followed by Hoechst fluorescence and propidium iodide analysis, indicated that small Lyt-2+ cells that are themselves noncycling were derived through mitosis from another cell population. The combination of the Hoechst-BrdU substitution technique with monoclonal antibodies and the organ culture should provide a powerful tool for the study of lineage pathways in the thymus.