Fukazawa H, Mizuno S, Uehara Y
Department of Bioactive Molecules, National Institute of Health, Tokyo, Japan.
Anal Biochem. 1995 Jun 10;228(1):83-90. doi: 10.1006/abio.1995.1318.
We developed a 96-well microplate assay to quantitate anchorage-independent growth of transformed cells. Wells of tissue culture microtiter plates are coated with poly(2-hydroxyethyl methacrylate) (poly-(HEMA)) to prevent cell attachment, and cells suspended in liquid media are seeded into the treated plates. Cell growth is assessed by tetrazolium dye reduction or by counting [3H]thymidine incorporation into DNA. There appeared to be a close correlation between growth in poly(HEMA)-coated plates and colony formation in soft agar, i.e., fibroblasts transformed by various oncogene proliferated in the coated plates, whereas their normal counterparts did not. Transformed cells on the nonadhesive surface were round and formed multicellular spheroids which resembled colonies in soft agar. Cells carrying either temperature-sensitive v-src or inducible v-Ki-ras oncogene proliferated on poly(HEMA)-coated plates only when they displayed transformed phenotype. This method is simple, quick, quantitative, and economical and in many cases might be more practical than conventional soft agar colony formation assay for measurement of anchorage-independent growth.
我们开发了一种96孔微孔板检测方法，用于定量转化细胞的非贴壁依赖性生长。组织培养微量滴定板的孔用聚（甲基丙烯酸2-羟乙酯）（聚-（HEMA））包被以防止细胞附着，然后将悬浮在液体培养基中的细胞接种到处理过的板中。通过四唑盐染料还原或通过计数掺入DNA中的[3H]胸苷来评估细胞生长。在聚（HEMA）包被的板中的生长与软琼脂中的集落形成之间似乎存在密切相关性，即被各种癌基因转化的成纤维细胞在包被的板中增殖，而它们的正常对应物则不增殖。在非粘附表面上的转化细胞呈圆形并形成多细胞球体，类似于软琼脂中的集落。携带温度敏感型v-src或可诱导型v-Ki-ras癌基因的细胞仅在显示转化表型时才在聚（HEMA）包被的板上增殖。该方法简单、快速、定量且经济，在许多情况下，对于测量非贴壁依赖性生长可能比传统的软琼脂集落形成检测更实用。
