8-azidoethidium and Necrosis

This work seeks to provide users with guidance on cell culture, treatment, processing and analytical conditions for achieving optimal performance of the in vitro micronucleus assay using the In Vitro MicroFlow(®) method. Experimental data are provided to support the advice described. The information provided covers specific topics or issues that are identified as critical to the methodology and thus is meant to work with instruction manuals, published papers and other references, and not as a replacement for these documents. The content is divided into several sections. Cell culture and treatment describes conditions for routine maintenance of cells as well as treatment with test articles. Preparation and processing of samples details steps found to be critical in execution of the procedure. Instrument parameters and analysis covers set-up of the flow cytometer and evaluation of the samples. General assay considerations and interpretation of results describes examination of data in terms of assay validity, viability and genotoxicity assessment. The goal is to educate users and enable them to design, conduct and interpret flow cytometric in vitro micronucleus (MN) studies. Readers should obtain an understanding of specific cell culture practices, options for assay formatting and execution and the information required to successfully integrate and validate the in vitro MN assay into their existing safety program.

Topics: Animals; Apoptosis; Azides; Cell Culture Techniques; Cells, Cultured; Cricetinae; DNA Damage; Flow Cytometry; Micronucleus Tests; Necrosis; Thymidine

The in vitro micronucleus test has received considerable attention in recent years for its use in drug safety assessment and toxicological research. The less tedious nature of the assay relative to chromosome aberration analyses is a driving force, and explains why many chemical and drug safety programs have adopted the endpoint. Development of a high-throughput micronucleus scoring system would further enhance the utility of the assay for lead optimization and other early drug development work. Although several variations of a flow cytometric (FCM) method for scoring cell-culture-derived micronuclei (MN) have been described in the literature, they have been unable to distinguish true MN from apoptotic and necrotic chromatin (Nüsse M and Marx K 1997: Mutat Res 392: 109-115). Here, we report advances to this methodology whereby a sequential staining procedure is used to differentially label these types of sub-2n particles. With the use of ethidium monoazide (EMA), the chromatin of dead and dying cells is labeled. After a photoactivation step that covalently binds EMA to chromatin, cytoplasmic membranes are digested and resulting lysates are incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and sub-2n particles that are labeled with either SYTOX or SYTOX and EMA. Preliminary studies with heat-shocked L5178Y mouse cells demonstrated that EMA stains necrotic and mid- through late-stage apoptotic cells. Importantly, the sequential labeling procedure provided reliable micronucleus enumeration, even when cultures contained high percentages of dead cells. Subsequently, experiments with the following diverse genotoxicants were performed: hydroxyurea, methyl methanesulfonate, benzo[a]pyrene, etoposide, cyclophosphamide, and vinblastine. Additionally, the nongenotoxicants sucrose, tributyltin methoxide, and dexamethasone were tested up to 5 mg/ml, or to cytotoxic concentrations. FCM data were found to correspond closely with microscopy-based measurements. Collectively, these data suggest that this sequential EMA/SYTOX staining procedure provides reliable, high-throughput enumeration of in vitro MN.

Topics: Animals; Apoptosis; Azides; Biological Assay; Cell Cycle; Cell Line, Tumor; Chromatin; Flow Cytometry; Fluorescent Dyes; Mice; Micronuclei, Chromosome-Defective; Micronucleus Tests; Microscopy, Fluorescence; Mutagens; Necrosis; Organic Chemicals; Reproducibility of Results