Flow Cytometric Analysis of Bursal, Thymic, and Splenic Cells from Normal and Cyclophosphamide-Treated Embryos

Abstract

Cell flow cytometry was used to assess the life cycle of embryonic lymphocytes. Cells were prepared for flow cytometry by fixation for 30 min in a final concentration of 70% ethyl alcohol, treated with ribonuclease (50 to 70 Kunitz/ml) and incubated for 30 min. in propidium iodide. Our data demonstrated that bursal lymphocytes from 20-day embryos (DE) exhibited significantly fewer cells in pre-DNA (deoxyribonucleic acid) and more cells in DNA synthesis (S) than thymic cells from 20 DE, and thymic lymphocytes from 16 DE expressed a more active S than bursal cells. Lymphocytes were separated from granulocytes by a Ficoll-hypaque double density gradient. The DNA cycle of bursal lymphocytes was more easily disrupted with cyclophosphamide (Cy) than the DNA cycle of thymic lymphocytes. However, unfractionated splenic cells from embryos treated with Cy revealed a greater percentage of cells in S and post-DNA synthesis and mitosis than splenic cells from control embryos. Because the splenic cells of Cy-treated embryos were predominantly immature granulocytes, it was concluded that Cy stimulated myelopoiesis in the spleen.