Coers W, Huitema S, Smeenk R J, Salant D J, Grond J, Weening J J
Department of Pathology, University of Groningen, The Netherlands.
Hybridoma. 1992 Aug;11(4):529-37. doi: 10.1089/hyb.1992.11.529.
A sensitive and reproducible microassay is described for quantification of adhesion of cells to matrix-coated 96-wells plates under different experimental conditions. For this purpose glomerular visceral epithelial cells (GVEC) were used. Attached GVEC were fixed with methanol and incubated with a monoclonal anti-DNA antibody. Following standard procedures, the amount of bound antibody was quantified by ELISA. A positive linear relationship in the range of 800-5000 cells per well was found between OD values and cell numbers obtained by hand-counting (r = 0.94, p less than 0.001). The assay is 10 to 100 times more sensitive than most other adhesion assays. The applicability of the ELISA assay was demonstrated by manipulation of the temperature during adhesion and by using different concentrations of the matrix-molecules fibronectin, EHS-laminin and collagen type I. The ELISA assay was found to be unaffected by non-specific interaction of anti-DNA antibodies with the matrix molecules used for coating. The assay was neither affected by potential release of DNA from the GVEC under these different experimental conditions. In conclusion, this cell adhesion microassay is simple, reliable, sensitive, and cost-effective, since it requires small amounts of GVEC and reagents.
本文描述了一种灵敏且可重复的微量测定法，用于在不同实验条件下定量细胞与基质包被的96孔板的黏附。为此使用了肾小球脏层上皮细胞（GVEC）。贴壁的GVEC用甲醇固定，并用单克隆抗DNA抗体孵育。按照标准程序，通过酶联免疫吸附测定（ELISA）对结合的抗体量进行定量。在每孔800 - 5000个细胞范围内，发现光密度（OD）值与手工计数获得的细胞数量之间呈正线性关系（r = 0.94，p < 0.001）。该测定法比大多数其他黏附测定法灵敏10至100倍。通过在黏附过程中控制温度以及使用不同浓度的基质分子纤连蛋白、EHS - 层粘连蛋白和I型胶原，证明了ELISA测定法的适用性。发现ELISA测定法不受抗DNA抗体与用于包被的基质分子的非特异性相互作用的影响。在这些不同实验条件下，该测定法也不受GVEC潜在释放的DNA的影响。总之，这种细胞黏附微量测定法简单、可靠、灵敏且具有成本效益，因为它需要少量的GVEC和试剂。
