Cell Cycle Assay
The Cell Cycle Assay is a high content imaging assay that reports the distribution of a cell population among G1, S, G2, and M phases of the cell cycle. Additionally, the presence of sub-G1 (fragmented or apoptotic) and super-G2 (clustered or polyploid) cells can be quantified. Results from this assay can detect and elucidate the nature of a proliferative arrest, acceleration, or deviation from the normal cycle.
In this assay, cells grown in microplates are treated with test articles for the desired duration. For 30 minutes prior to fixation, cells are incubated with EdU, a thymidine analog, which is taken into cells and incorporated into the DNA of those that are replicating their genome as part of S-phase. Cells are then fixed, and the EdU in S-phase cells is labeled using click chemistry. The Cell Cycle Assay can be multiplexed with additional measurements, such as cell counts or nuclear morphology, or with immunofluorescent detection of an additional protein of interest.
Cell Cycle Assay. (A) Cells cycle through G1, S, G2, and M phases. EdU incorporation is a marker of S-phase and phosphor-Histone H3 is a marker of M-phase. (B) HCT116 cells stained for DNA with DAPI (blue), EdU (green), and phospho(ser10) Histone H3 (red). (C) A scatterplot of cells showing the EdU and DAPI labeling intensities of cell sub-populations in different phases of the cell cycle.
Cell Cycle Assay example. In this experiment, a cancer cell line was treated with a dose range of test article for 24 h, and then cell cycle distribution was determined for each sample. (M-phase was not assessed in this example). Points and error bars are means and standard deviations of triplicate samples. The results show a dose-dependent increase in G1 cells with concomitant decrease of cells in S and G2 phases.