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 hypoxanthineguanine-phosphoribosyl-transferase. Flow cytometric sorting of proliferating cells in the late S-and the Gz-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 G,/G* 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 Gz-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.
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