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Quantitative Analysis of the Cell Cycle in Imaging Applications

ination to specifically inform on the M and S phases of the cell cycle while it is still possible to drop in an additional specificity of interest to the user. For this assay, Hoechst 33342 was used to identify all nuclei and an anti-phospho-Histone 3 antibody was used to identify cells in M phase.1 Similarly immunofluorescent staining of incorporated bromodeoxyuridine (BrdU) is used to determine the frequency of cells that had synthesized DNA.2, 3, 4, 5 In this method, BrdU (an analog of the DNA precursor thymidine) is incorporated into newly synthesized DNA by cells entering into and progressing through the S phase of the cell cycle. The drop in choice for this assay was a fluorescently labeled anti-Ki-67 antibody. Ki-67 is present in all cycling cells but is not present in quiescent cells (G0). The Ki-67 staining pattern is variable depending on the phase of the cycle, in the G1/S/G2 phases it is punctuate, whereas in late G2/M it appears throughout the entire nucleus (see Table 1 ).6,7

The BD Pathway Bioimager is well suited for the development of complex assays due to its wide range of acquisition and analysis tools. Using a broad-spectrum illumination source, the system enables the user to select the antibodies based on their compatibility with the assay rather than with specific laser wavelengths. The 16 excitation filter choices of the BD Pathway Bioimager allow the setup of several multi-color assays with minimal bleed-through which enables the system to be used in a wide variety of demanding applications.

Methods

HeLa cells (a human cervical cancer cell line, ATCC, #CCL-2) were seeded in 96-well clear bottom tissue culture plates optimized for automated imaging applications (Cat. No. 35-3219) at 15,000 cells/well in DMEM (Invitrogen, 11965-084) complete media. Eighteen hours later cells were treated with one of two different metabolic inhibitors, aphidicolin (Sigma-Aldric
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