Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis
Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis
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DOI：
10.1002/cyto.10149
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发表时间：
2002-10-01
期刊：
影响因子：
--
通讯作者：
Campisi, J
中科院分区：
文献类型：
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作者：
Krtolica, A;de Solorzano, CO;Campisi, J
背景：为了证明衰老成纤维细胞刺激癌前上皮细胞的增殖和肿瘤转化（Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001），我们开发了量化与成纤维细胞共培养的上皮细胞增殖的方法。方法：用荧光dna嵌入染料4,6-二氨基苯基酚（DAPI）对上皮-成纤维细胞共培养物进行染色，或在上皮细胞中表达绿色荧光蛋白（GFP），然后与成纤维细胞一起培养。用合适的滤镜在倒置显微镜下拍摄共培养物，并用数码相机捕捉荧光图像。我们修改了一个图像分析程序，以选择性地识别DAPI染色培养物中更小、荧光更强烈的上皮细胞核，并使用该程序量化每个领域中上皮细胞核产生DAPI荧光或上皮细胞产生GFP荧光的区域。结果：用DAPI和GFP荧光分析图像区域产生了几乎相同的上皮细胞与成纤维细胞共培养的定量。我们通过人工计数限制了这些结果。此外，GFP标记允许在多个时间点上对同一共培养进行动力学研究。结论：我们描述的基于图像分析的定量方法是一种简单可靠的共培养细胞监测方法，可用于各种细胞生物学研究。(C) 2002 Wiley-Liss, Inc。
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-diamidino2-phenyhndole (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 confinned 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. (C) 2002 Wiley-Liss, Inc.