Direct and indirect flow cytometric enumeration of 6-thioguanine-resistant human peripheral blood lymphocytes.
Methods based on flow cytometry and sorting, autoradiography, and cloning were used to evaluate the potential for the enumeration of 6-thioguanine-resistant human peripheral blood lymphocytes assumed to be deficient with respect to the enzyme hypoxanthine-guanine-phosphoribosyl-transferase. Flow cytometric sorting of proliferating cells in the late S- and the G2-stages by means of DNA content, as measured by propidium iodide fluorescence, enabled an enrichment of variant cells to about 99%. The main source of false events was contaminating doublets of G0/G1 cells appearing in the sorting region. Doublet discrimination measured as the difference between pulse height and area (Ortho-50) accomplished no further improvement. A combination of propidium iodide fluorescence and bromodeoxyuridine incorporation, measured by fluorescent anti-bromodeoxyuridine-DNA antibodies, allowed flow cytometric enrichment to about 99.99% of variant cells. By sorting of 3H-thymidine-labeled cell nuclei from the late S- and the G2-phases and subsequent autoradiographic evaluation, partly resistant variants could be discriminated; variant frequencies of the same magnitude as for the cell cloning methods were obtained.
D000911:Antibodies, Monoclonal; D001345:Autoradiography; D001973:Bromodeoxyuridine; D002469:Cell Separation; D002470:Cell Survival; D004247:DNA / Q000032:analysis; D004351:Drug Resistance; D005434:Flow Cytometry; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000172:deficiency*; D008214:Lymphocytes / Q000187:drug effects* / Q000201:enzymology; D011419:Propidium; D013866:Thioguanine / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D011419:Propidium; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine; D001973:Bromodeoxyuridine