Evaluation of the Litron In Vitro MicroFlow ® Kit for the flow cytometric enumeration of micronuclei (MN) in mammalian cells

Abstract

We have evaluated the performance of the prototype In Vitro MicroFlow ® Kit (Litron Laboratories), which offers a flow cytometric method for scoring micronuclei (MN). This method uses sequential staining to differentiate MN from chromatin fragments derived from apoptotic or necrotic cells. Data were generated using the genotoxins methylmethane sulphonate (MMS), dimethylbenzanthracene (DMBA) and vinblastine, and the non-genotoxins dexamethasone and staurosporine, which are known to induce apoptosis in vitro. The results obtained with these agents were compared with conventional microscopy. For short-duration exposures (3–4 h) both manual and flow methodologies demonstrated good concordance, with concentration-related increases in the percentage of MN for MMS, DMBA and vinblastine. Statistically significant increases were observed at ≥20 and 40 μg/mL, for manual and flow analysis, respectively, for MMS; at 0.5 and 0.75 μg/mL for DMBA; and at 0.035 and 0.04 μg/mL, respectively, for vinblastine. Dexamethasone showed clear negative responses by manual and flow cytometric analysis, with comparable results for both methodologies (all <1.7-fold compared with concurrent vehicle controls). Data for staurosporine, however, were less consistent showing significantly higher flow cytometric MN frequencies compared with those seen after manual analysis. Continuous (24 h) treatments were also conducted with MMS, vinblastine, dexamethasone and staurosporine. There was good concordance between the methodologies for MMS, staurosporine and vinblastine. However, dexamethasone generated discordant results, i.e. microscopic analysis was clearly negative at all doses tested, whereas flow cytometry produced significant increases in MN frequency (up to 8.1-fold at 100 μg/mL compared with the concurrent vehicle control). The inconsistencies observed between flow cytometry and standard microscopy, and the differences in assay sensitivity, particularly for apoptosis-inducing compounds, suggest that the prototype In Vitro MicroFlow ® Kit requires further refinement. Studies to investigate new parameters to address these issues are now under way and will be reported separately.