Along with micronucleus measurements, this automated, flow cytometer-based assay has the benefit of providing the user with multiple endpoints. These endpoints can help to better characterize your lead compounds with more confidence, and include:

Micronucleus Formation: Micronuclei contain small amounts of nucleic acid (DNA) and thus can be tracked with dyes that specifically label nucleic acids.  By employing our sequential staining and lysis procedure, nuclei, micronuclei and nuclear fragments are liberated from the cell and differentiated by the flow cytometer based on their size and dye uptake characteristics.         

Cytotoxicity Assessment: Pilot experiments evaluated several cytotoxicity endpoints, including Coulter Counts, total DNA content, and an ATP-based plate reader assay. However, these assays tended to underestimate toxicity relative to cell counts that were performed with a vital stain. In order to provide a more complete assessment of cytotoxicity, we employ 3 flow cytometry-based measurements that are not only compatible with the MN scoring application, but are acquired simultaneously with MN scoring.

• Relative Survival: 6 micron orange fluorescent latex beads are at various points throughout culturing or treatment.  By analyzing these “counting beads” on the flow cytometer, the number of healthy (EMA-negative) nuclei per bead can be determined and a Nuclei-to-Bead ratio calculated. This measurement is analogous to relative cell count, but with the advantage that an assessment of cell membrane integrity is made.

• % Dead/Dying Cells: The percentage of dead/dying cells (i.e. EMA-positive events) is acquired simultaneously with those above.  Experiments to date suggest interpretation of MN values in samples displaying a level of EMA+ events beyond a certain criteria should be considered with extreme caution.

• Cell Cycle Information: Test article-induced cell cycle effects can be analyzed (derived from a histogram expressing linear SYTOX fluorescence, not shown). For instance, the G2/M block associated with MMS treatment is readily apparent. Dose-dependent staurosporine cell cycle effects are presented in Figure 4.

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