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 w ith 6 Gy for characterization of their DNA content, using gel electrop horesis. A novel method for isolation of MNC based on a sucrose gradie nt ultracentrifugation was developed. With this efficient method (80% recovery) more than 150 x 10(6) MNC per day could be isolated. The pur ity was > 99%. However, the low number of main nuclei in the MNC isola te (< 1%) resulted in a contamination of MNC DNA with about 15% main n ucleus DNA, due to the several times higher DNA content of main nuclei . Cell sorting was utilized to maximize the purity by choosing the rec ommended sorting mode for highest purity. Isolation of MNC with the ce ll sorter was successful (100% purity), but also time-consuming (1-2 x 10(6) MNC per working day could be isolated) and insufficient (10% re covery). 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 f or 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 isolatio n of large amounts of purified (> 99%) MNC without the employment of s ophisticated and expensive techniques (cell sorting) and the accompani ed knowhow. In contrast, maximum purity but low yields of MNC can be o btained by cell sorting.