A modified protocol for the cytochalasin B in vitro micronucleus assay using whole human blood or separated lymphocyte cultures

Abstract

A modified protocol is described for the in vitro analysis of micronuclei in whole blood or separated lymphocyte cultures. The induction of binucleate cells by various concentrations of cytochalasin B (3, 4.5 or 6 micrograms/ml) was examined at two harvest times (68 or 72 h). An optimal yield was obtained by adding cytochalasin B at a dose of 6 micrograms/ml to cultures 44 h after initiation with harvest 24 h (whole blood) or 28 h (separated lymphocytes) later. Cytocentrifuge preparations of lymphocytes (separated from whole blood using commercial preparations of Ficoll either at the commencement of the assay or upon harvest) were stained with Acridine Orange. Using this method, cytokinesis-blocked lymphocytes remain intact and micronuclei are readily identified. The method is suitable for both whole blood and separated lymphocyte cultures, thus allowing direct comparisons of sensitivity to genotoxic agents.