( protocol 1. Seed CHO-K1 cel...)Analysis of in vitro micronucleus assays using the IN Cell Analyzer 3000 Micronuclei Formation Analysis Module

2. Prepare dilutions of test compounds in solvent. Prepare a 1-mg/ml stock solution of mitomycin C in PBS and sterile filter for use as a positive control. Add 100 μl of test compounds, solvent, and mitomycin C (use mitomycin C at 100-ng/ml final concentration) and incubate cells for 24 h.

3. Prepare a 3-mg/ml stock solution of cytochalasin B in DMSO. Dilute 1:100 in complete tissue culture medium and add 10-μl/well. Incubate cells for 24 h.

4. Remove media, wash cells once with PBS, and fix in ethanol for 30 min at room temperature.

5. Make 10-mg/ml stock solution of FITC in DMSO. Dilute 10 μl in 100-ml PBS and add 100-μl/well. Incubate for 30 min at room temperature.

6. Wash wells three times with PBS.

7. Stain cells with 5-μM Hoechst™ 33342 in PBS for 15 min at room temperature.

8. Image wells using excitation and emission settings for fluorescein and Hoechst 33342.

Analysis protocol
The IN Cell Analyzer 3000 Micronuclei Formation Analysis Module uses a series of operations based on user settings (Fig 2) to define nuclei, segregate mono-nucleate and binucleate cells, and define a search area for micronuclei (Fig 3). For full guidance on using the analysis module see the product manual.

1. Open run file in the IN Cell Analyzer 3000 analysis software.

2. Select a mitomycin C positive control well. Apply intensity threshold, erosion, and size filtration settings to identify nuclei in the blue (Hoechst) channel. Take care to exclude micronuclei from identified objects.

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