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Identification of cells in S phase using the Cell Proliferation Fluorescence Kit and IN Cell Analyzer 1000

scribed in the Method section. Plates were imaged using the IN Cell Analyzer 1000. Figure 1 depicts nuclei of U2OS cells stained with Hoechst nuclear dye (blue). BrdU incorporation is shown in pink (Cy5). Cells in S phase are identified by their pink nuclei.

Figure 2 depicts the effect of Mitomycin C (MMC), a cytostatic drug that inhibits mitosis causing cells to accumulate in G2 phase of the cell cycle. Cells were incubated with increasing concentrations of MMC (as indicated below each set of triplicate images) for 18 h in a 96-well microplate.

Figure 3 is a graphic representation of the fluorescence intensity of each nucleus from the two wells (indicated by arrows) in Figure 2. The Object Intensity Analysis Module was used to derive the nuclear fluorescence intensity (IPos) for each cell (Cell by cell analysis setting). Each data point represents the nuclear fluorescence intensity of one cell. Approximately 250 cells were analyzed per analysis field in each well.

Figure 4 depicts pie charts representing the percentage of cells with a nuclear fluorescence intensity above 300 (these are considered to be in S phase), calculated from data used to plot Figure 3.

Figure 5 shows dose response representing the effect of MMC on the average nuclear fluorescence intensity per well. This was derived using the Object Intensity Analysis Module (Summary setting). Each data point represents the average nuclear fluorescence intensity of three wells. Approximately 250 cells were analyzed per analysis field in each well.


Conclusion
The Cell Proliferation Fluorescence Kit is a sensitive and robust assay that can be used in conjunction with the IN Cell Analyzer 1000 in a fixed-cell assay format to identify cells that are in S phase (Fig 2) and also
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