Fenech M, Morley A
Cytobios. 1985;43(172-173):233-46.
The standard micronucleus assay as applied to lymphocyte cultures does not discriminate between non-dividing cells and cells that have divided. Micronuclei can only be expressed after nuclear division and because the proportion of dividing cells may vary from one culture to another the standard micronucleus assay can be very imprecise. To overcome this problem we have developed four new methods for scoring micronuclei only in lymphocytes that have divided once only. In the stathmokinetic method the proportion of dividing cells was measured by blocking them at metaphase with colchicine and the number of micronuclei was determined in a parallel culture. The ratio of micronucleus index and metaphase index gave the number of micronuclei per metaphase. With the flow cytometric method, the cells in S phase were allowed to incorporate bromodeoxyuridine and then allowed to divide. By virtue of their reduced fluorescence after staining with Hoechst 33342 it was possible to sort these cells out with a fluorescence activated cell sorter. The autoradiographic method employed incorporation of tritiated thymidine in cells in S phase so that after division these cells could be recognised by their labelled nuclei. In the cytokinesis-block method, dividing cells were inhibited from performing cytokinesis by exposing them to 3.0 micrograms/ml cytochalasin-B. Dividing cells were easily recognised by their binucleate appearance. Results from the four methods for micronuclei induced by X-rays were in close agreement, but the simplest and most precise of the methods was the cytokinesis-block method.
应用于淋巴细胞培养的标准微核试验无法区分未分裂细胞和已分裂细胞。微核只有在核分裂后才能显现，而且由于不同培养物中分裂细胞的比例可能不同，标准微核试验可能非常不精确。为克服这一问题，我们开发了四种新方法，仅对仅分裂过一次的淋巴细胞中的微核进行计数。在有丝分裂抑制法中，通过用秋水仙碱将细胞阻断在中期来测量分裂细胞的比例，并在平行培养物中测定微核数量。微核指数与中期指数的比值给出了每个中期的微核数量。采用流式细胞术方法时，让处于S期的细胞掺入溴脱氧尿苷，然后使其分裂。由于用Hoechst 33342染色后其荧光减弱，可用荧光激活细胞分选仪将这些细胞分选出来。放射自显影法采用将氚标记的胸腺嘧啶核苷掺入处于S期的细胞中，这样在细胞分裂后，这些细胞可通过其标记的细胞核被识别。在胞质分裂阻断法中，将细胞暴露于3.0微克/毫升的细胞松弛素B中，抑制分裂细胞进行胞质分裂。分裂细胞因其双核外观很容易被识别。四种方法检测X射线诱导的微核的结果非常一致，但其中最简单、最精确的方法是胞质分裂阻断法。
