Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.
BACKGROUND
To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts.


METHODS
We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field.


RESULTS
Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points.


CONCLUSIONS
The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies.
D002455:Cell Division; D002467:Cell Nucleus; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D018920:Coculture Techniques; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D019044:Image Cytometry / Q000295:instrumentation / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D007211:Indoles; D008164:Luminescent Proteins / Q000378:metabolism; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D007211:Indoles; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; C007293:DAPI