Isolation of micronuclei: high yield by sucrose gradient versus maximum purity by cell sorting.
Micronuclei (MNC) of L929 cells were isolated 72 h after irradiation with 6 Gy for characterization of their DNA content, using gel electrophoresis. A novel method for isolation of MNC based on a sucrose gradient ultracentrifugation was developed. With this efficient method (80% recovery) more than 150 x 10(6) MNC per day could be isolated. The purity was > 99%. However, the low number of main nuclei in the MNC isolate ( < 1%) resulted in a contamination of MNC DNA with about 15% main nucleus DNA, due to the several times higher DNA content of main nuclei. Cell sorting was utilized to maximize the purity by choosing the recommended sorting mode for highest purity. Isolation of MNC with the cell sorter was successful (100% purity), but also time-consuming (1-2 x 10(6) MNC per working day could be isolated) and insufficient (10% recovery). Extraction of DNA of these isolated MNC resulted in less than 1 ng/day. Hence, at least 1 week of cell sorting would be necessary for one electrophoretic run. When employing the sucrose gradient method, 2000 times more DNA of MNC have been isolated. We therefore consider this method as the most efficient way for rapid and low cost isolation of large amounts of purified ( > 99%) MNC without the employment of sophisticated and expensive techniques (cell sorting) and the accompanied knowhow. In contrast, maximum purity but low yields of MNC can be obtained by cell sorting.
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Institute of Radiobiology, Federal Armed Forces Medical Academy, Munich, Germany 100305.3537@compuserve.com
Frombeck
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Baaske
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van Beuningen
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D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000302:isolation & purification; D004996:Ethidium / Q000378:metabolism; D005354:Fibrosarcoma; D005434:Flow Cytometry; D051379:Mice; D048629:Micronuclei, Chromosome-Defective / Q000737:chemistry; D014407:Tumor Cells, Cultured
D004247:DNA; D004996:Ethidium