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Quantitative Analysis of the Cell Cycle in Imaging Applications

The staining pattern for BrdU and phospho-Histone H3 found in control cells was consistent with a heterogeneous cell population ( Figure 1 ). We found that approximately 3% were in M phase based on the positive phospho-Histone 3 staining and approximately 22% were in S phase based on the BrdU results ( Figure 1D ). When cells were treated with either colcemid or aphidicolin changes in cell cycle status were seen ( Figure 1 and Figure 3 ). In colcemid treated cells (eg, 500 ng/mL) the percent cells in S phase remained similar (~20%) while the percent cells in M phase doubled to approximately 7% ( Figure 1D ). In aphidicolin treated cells (eg, 500ng/mL), the percent cells in S phase dropped to 0% while the percent cells in M phase dropped slightly to approximately 2%. Figure 2 shows the dose-response relationship of BrdU incorporation in response to increasing concentrations of colcemid and aphidicolin. BrdU indicates cells in S phase and colcemid had no significant effect at any concentration. Aphidicolin decreased the number of S-phase cells in a dose-dependent manner.

Discussion

To assure homogenous responses in cellular assays, cells are often synchronized using multiple rounds of cell-cycle arrest and release. This adds complexity and unreliability to the assay in question and is incompatible with many assays especially stem cell or primary cell assays. We therefore developed a versatile application that utilizes standard antibody treatment to identify the cell cycle phases and applied it to automated imaging. As a stand-alone application, the assay quantifies the degree to which cells are arrested in the different cycle phases after drug treatment making it useful in oncology and other fields where drug-responses are suspected to be dependent on the specific cell cycle phase. In addition, the nature of the exchangeable antibodies allow
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