A Double Fluorescent Staining Method Increases the Sensitivity of the Cytokinesis-Block Micronucleus Assay

The micronucleus test is one of the most popular genotoxicity assays. In order to avoid underestimation of micronuclei frequencies by counting non-replicating cells, the cytokinesis-blocked micronucleus test has been developed. In this technique, only binucleated cells are scored. One underestimated problem is the potential difficulty in discriminating binucleated from mononucleated cells when using DAPI staining, i.e., the possibility that two neighboring mononucleated cells could be mistaken for a binucleated one. The new protocol presented here comprises the addition of acridine orange in order to stain the cytoplasm (in addition to DAPI to stain nuclei and micronuclei). This new technique can increase the sensitivity of the cytokinesis-blocked micronucleus test and avoid underestimation of micronuclei frequencies, an important issue when high doses are employed.