Comparison of antigen contents in co-cultures by an in situ immunoradiometric assay

A fast, sensitive and reproducible in situ immunoradiometric assay has been developed to compare relative contents of cellular markers in cultures. Th is assay is performed directly in the multi-well plate. After methanol fixa tion, antigens are identified by specific primary antibodies, followed by I -125-protein A. Cell-associated radioactivity is then measured in lysates u sing a gamma radiation counter and expressed with respect to protein conten t. By this method, differences in the level of any antigen retained by fixa tion can be easily quantified. The convenience, dynamic range of linearity and reproducibility of this technique compare favorably with Western blotti ng. Originally, the assay was designed to monitor the relative abundance of glial or neuronal cells in embryonic cerebral cocultures upon various expe rimental conditions, by measuring related changes in glial fibrillary acidi c protein (GFAP) or microtubule-associated protein 2 (MAP-2) content. It is proposed as a method of choice to quantify the effects of culture conditio ns or toxic agents on a specific cell type in mixed populations. (C) 1999 E ditions scientifiques et medicales Elsevier SAS.