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首页> 外文期刊>Journal of Neuroscience Methods >Use of cell ELISA for the screening of neurotrophic activities on minor cell populations in retinal monolayer cultures.
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Use of cell ELISA for the screening of neurotrophic activities on minor cell populations in retinal monolayer cultures.

机译：细胞ELISA用于筛选视网膜单层培养物中次要细胞群体的神经营养活性。

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In this study we describe a large-scale screening cell ELISA protocol which is suitable for the characterization of exogenic factor effects in mixed central nervous system (CNS) culture. The main novelty of the assay is that it permits the measurement of cellular responses in populations comprising as little as 2-4% of the total cell number. For standardization of the assay, we employed antibodies against opsin and microtubule-associated protein (MAP2) which label distinct retinal cell classes. Embryonic chick retinal neurons were grown in microtiter plates and directly processed for detection of antibody binding on the same plate. Binding of the antibodies was saturable and the ELISA signal was proportional to the number of immunoreactive cells comprising 2-4% and 16% of the total cell number with opsin and MAP2 antibodies, respectively. A minimum of 2000 opsin-positive cells could be reliably determined. Using our cell ELISA protocol, we demonstrate a developmental increase of both cell markers whichreflected an increase in the number of opsin-positive cells but an enhanced expression per cell in the case of MAP2. We also show that growth-promoting activity-the presumed chick ciliary neurotrophic factor (CNTF)-stimulated the expression of opsin in retinal cultures (EC50; 2.3 pM) and that a corresponding activity is specifically expressed in the developing retina. Our results show that the cell ELISA protocol allows the rapid screening for distinct, low-percentage cell populations responding to exogenous factors in mixed CNS cultures.
机译：在这项研究中，我们描述了一种大规模筛选细胞ELISA方案，适用于表征混合中枢神经系统（CNS）培养物中外源因子的作用。该测定法的主要新颖之处在于它可以测量仅占总细胞数2-4％的群体的细胞反应。为了标准化分析，我们使用了针对视蛋白和微管相关蛋白（MAP2）的抗体，它们标记了不同的视网膜细胞类型。胚胎小鸡视网膜神经元在微量滴定板中生长，并直接处理以检测同一板上的抗体结合。抗体的结合是饱和的，并且ELISA信号与视蛋白和MAP2抗体分别与构成总细胞数的2-4％和16％的免疫反应细胞的数量成比例。至少可以确定2000个视蛋白阳性细胞。使用我们的细胞ELISA方案，我们证明了两种细胞标志物的发育增加，这反映了视蛋白阳性细胞数量的增加，但在MAP2的情况下，每个细胞的表达增加。我们还显示，促进生长的活性（假定的雏鸡睫状神经营养因子（CNTF））刺激了视黄醛在视网膜培养物中的表达（EC50; 2.3 pM），并且相应的活性在发育中的视网膜中被特异性表达。我们的结果表明，细胞ELISA方案可以快速筛选对混合的CNS培养物中的外源因子有反应的不同的低百分比细胞群。

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