Application of cell sorting for enhancing the performance of the cytokinesis-block micronucleus assay

Abstract

Among the numerous methods available to assess genotoxicity, the cytokinesis-block micronucleus (CBMN) assay is very popular due its relative simplicity and power to detect both clastogenic and aneugenic compounds. A problem with the CBMN assay is that all DNA damaging agents also inhibit the ability of cells to progress through mitosis, leading to a low number of binucleated cells (BNCs). One method to resolve this issue is to ensure a sufficient proportion of BNCs in the samples. In the current study, the applicability of a cell sorting system capable of isolating cell fractions containing abundant BNCs was investigated. Furthermore, to investigate the relationship between the cell division delay due to radiation exposure and the generation of BNCs and micronuclei (MN), we assessed a series of lag times between radiation exposure and addition of cytochalasin-B (Cyt-B). Cells from the human chronic myelogenous leukemia cell line K562 were exposed to X-rays (2 Gy and 4 Gy), and Cyt-B was subsequently added at 0, 6 and 12 h following irradiation. After treatment with Cyt-B for 24 h, the percentage of BNCs, the MN frequency and the cell cycle distribution were analyzed. In addition, cells displaying the DNA contents corresponding to BNCs were isolated and analyzed. The results indicate that applying the cell sorter to the CBMN assay increased the percentage of BNCs compared with the standard method. Thus, this technique is a promising way of enhancing the capacity of the CBMN assay.