This automated, flow cytometer-based assay has the benefit of providing the user with several valuable endpoints that provide a more comprehensive characterization of treatment-related toxicity.

Genetic Toxicity Assessment

• % Micronucleus Formation: Micronucleus frequency provides information on a chemical’s potential to induce chromosome damage.
• Mode of Action: When used with certain cell lines (e.g. hamster CHO-K1), the percentage of hypodiploid nuclei can provide information regarding mode of action, discriminating aneugens from clastogens.

Cytotoxicity Assessment: In order to provide a comprehensive assessment of cytotoxicity, we employ three flow cytometry-based measurements that are acquired simultaneously with MN scoring. 

• Cell Number: 6 micron orange fluorescent latex beads may be included at one of several points during the assay.  By scoring these ‘counting beads’ on the flow cytometer, relative survival, relative increased cell counts and relative population doublings can be determined.  These measurements are useful for setting top concentration of test article.
• Dead/Dying Cells: The health of treated cells can be inferred from the percentage of particles that are stained with Nucleic Acid Dye A (EMA-positive).  Since the fragmented nuclei of apoptotic cells can each form many such particles, this statistic is particularly sensitive to test article-induced apoptosis.  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 perturbations to the cell cycle are apparent from DNA-associated fluorescence measurements. For instance, the G2/M block associated with MMS treatment is readily apparent. Dose-dependent staurosporine cell cycle effects are presented in Figure 4.