The induction of micronuclei is a key characteristic of genotoxic compounds; for example, clastrogens produce DNA strand breakage while aneugens interfere with chromosome segregation. As a result, assays that detect micronuclei have become an important component of toxicology screening for new drug candidates.
Traditionally, micronucleus screening was performed manually by highly skilled technicians using optical microscopy and cell counting; with this time-consuming method, however, analysis was subject to operator variance, bias, and error. An improved alternative is now provided with GE Healthcare’s IN Cell Analyzer 2000 high-content analysis system. The sub-cellular resolution of the system enables micronuclei to be easily discerned, while the automated software provides faster analysis and consistent objective scoring.
Guidelines for in vitro micronucleus assays used in genotoxicity testing usually recommend scoring at least 2,000 cells per treatment condition for single samples, or at least 1,000 cells per condition if replicate assays are used. Therefore, a critical factor in establishing robust high-content cell assays is the assurance that sufficient cells are imaged per treatment condition.
This is attained on IN Cell Analyzer 2000 by automatically counting cells “online” as each image is acquired. In this optional acquisition mode, successive fields of view are acquired until a preset cell-count threshold is achieved.
Online cell counting has the additional advantage of reducing plate read times and the data-storage burden as no excess images are acquired once the required number of cells has been imaged.
Variable Cell Counts
For the purposes of this tutorial, CHO-K1 cells were seeded onto 96-well Matriplate microplates and incubated with mitomycin C (clastogen) or etoposide (aneugen) for 48 hours to induce micronuclei formation. The cells were fixed with ethanol and stained with FITC and Hoechst 33342. With the online cell-count threshold set to 1,000 cells/well, plates were imaged on IN Cell Analyzer 2000 with the 20x/0.45 NA objective.
The improved performance of the online cell-counting feature in variable cell-count assays is illustrated (Table 1) compared to results obtained using an offline analysis protocol. For mononucleated micronucleus assays configured with multiple replicates per treatment condition, a minimum cell count of 1,000 cells/well is recommended.
Without online cell counting, 34 fields of view needed to be acquired from each well to ensure a sufficient cell count was achieved (based on the maximum field count obtained when online cell counting was applied).
This resulted in a plate acquisition time of 80 minutes with the standard-chip camera, compared to an acquistion time of 37 minutes when online cell counting was applied under comparable conditions. As summarized in Table 1, the use of online cell counting significantly decreased the plate acquisition times for the assay.