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October 01, 2010 (Vol. 30, No. 17)

Improving Assays for Micronuclei Detection

Application of High-Content Analysis System Can Be Beneficial in Toxicity Screening

  • Dose-Response Assays

    Click Image To Enlarge +
    Figure 2. Dose-response micronuclei formation assays: CHO K1 cells were treated with increasing doses of (A) etoposide or (B) mitomycin C and imaged on IN Cell Analyzer 2000 using the large-chip camera. Data points represent mean ±1 SEM, n=8 wells per concentration. Percentage maximum cell count was calculated as [100 x (mean field cell count of treated sample)/(mean field cell count of untreated control sample)].

    For dose-response assays, percentage maximum cell count and percentage of cells with micronuclei were plotted against drug concentration (Figure 2). Results show that micronuclei count increases with the drug treatment until toxic doses are reached and cells begin to detach from the plate.

    Percentage maximum cell count relative to untreated controls can be used as a crude index of cell proliferation (PI), which typically decreases with increasing exposure to genotoxins.

    Drug concentration at half maximal proliferation (PI50) was determined to be 127 nM for mitomycin C and 176 nM for etoposide (Table 2). These values are within the 95% confidence intervals of values obtained with the standard-chip camera, and correspond well with those reported in the literature (198 and 216 nM respectively) during validation of an automated micronucleus screening assay using GE Healthcare’s IN Cell Analyzer 1000.

  • Click Image To Enlarge +
    Table 2. Dose-response micronuclei formation assays imaged with the large and standard chip cameras

    The percentage of the cells presenting with micronuclei at PI50 was also determined for each assay. Table 2 shows that values obtained with the standard- and large-chip camera were comparable to each other as well as to previously reported values.

  • Conclusions

    The automatic online cell-counting facility of the IN Cell Analyzer 2000 enables the minimum number of cells required for statistical relevance to be imaged for each micronucleus assay. With this optional acquisition mode, successive fields of view are acquired until a preset count threshold is achieved.

    For micronucleus assays, online cell counting ensures that the minimum cell count is reliably achieved even when test compound toxicity results in a cell-count decrease. Online cell counting has the additional advantage of reducing plate read times and the data-storage burden, since no excess images are acquired once the desired number of cells has been imaged. Furthermore, use of the large-chip camera option enables even faster results, with plate read times reduced by as much as 50%.

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