A flow-cytometric assay is described that can be used to determine the frequency and the DNA content of micronucleated polychromatic (PCE) and normochromatic (NCE) erythrocytes in mouse peripheral blood. Thiazole orange was used for discrimination between PCEs and NCEs, while Hoechst 33342 was used to detect micronucleated PCEs and NCEs. Up to 70,000 polychromatic erythrocytes can be analyzed in less than 10 min. This corresponds to 1NL3,OoO micronucleated polychromatic erythrocytes, 90-95% of which are true events as determined with a fluorescence microscope after sorting. Using X-rays as the inducing agent in dose-response experiments, a significant increase can be registered at doses of 0.02 Gy. It seems possible that the method will also allow the detection of clastogenic effects of other inducing agents at lower doses than previously possible. o 1992 Wiey-Liss, I~C .

The frequencies and DNA distributions of micronuclei (MN) in polychromatic erthrocytes (PCEs) from bone marrow (BM) and peripheral blood (PB) of mice after four different treatments were determined by flow cytometry. PCEs were differentiated from normochromatic erythrocytes (NCEs) using the fluorescent RNA stain Thiazole orange, while MN in erythrocytes were detected with the DNA stain Hoechst 33342. The treatments were X-irradiation (1 Gy), cyclophosphamide (CPA; 30 mg/kg), vincristine sulphate (VCR; 0.08 mg/kg), and cholchicine (COL; 1 mg/kg). All treatments showed increased frequencies of micronucleated PCEs at 30 h after treatment in BM and at 50 h in PB. The clastogens (X-irradiation and CPA) and the spindle poisons (VCR and COL) could be grouped according to the fluorescent characteristics of the induced MN as well as the relative frequency of small (0.5-2% of the diploid DNA content) and large (2-10%) MN. In PB the relative frequency of large MN was lower than in BM, indicating that they were partly eliminated before entrance into the peripheral circulation.

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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.