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Analysis of in vitro micronucleus assays using the IN Cell Analyzer 3000 Micronuclei Formation Analysis Module

. Adjust nuclear power (DNA content) and form factor (nuclear symmetry) settings to segregate mono-nucleate and bi-nucleate cells. Use delta threshold settings if required to assign ambiguous cells as unclassified.

4. Adjust threshold in the green (FITC) channel to identify cell boundaries.

5. Adjust search area settings to define regions to be analyzed for micronuclei.

6. Adjust intensity and size filtration settings to identify micronuclei.

7. Check analysis on second positive control well and solvent negative control wells. Adjust analysis parameters as necessary.

8. Run analysis.


Data analysis
1. Open the data output file and analyze micronuclei frequency (Fig 4) and proliferation index (Fig 5) data.

2. Discard data from wells where the cell number or the proliferation index indicate cytotoxic or cytostatic activity. Compounds showing potential false negative criteria should be re-tested at a lower concentration.

Data from typical assays carried out using the protocol described are shown in Figure 4. Exposure of cells to increasing concentrations of compounds of known genotoxicity results in an increase in the percentage of bi-nucleate cells with micronuclei. As cells are exposed to higher doses of compounds, cell cycle inhibition and cytotoxicity results in cell arrest prior to mitosis preventing micronuclei formation, with a resulting drop in micronuclei frequency at higher compound doses. This is evident from examination of proliferation index data (Fig 5), which reports the ratio of bi-nucleate to mono-nucleate cells.

Analysis of micronucleus assay data
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