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. This assay is performed directly in the multi-well plate. After methanol fixation, antigens are identified by specific primary antibodies, followed by 125I-protein A. Cell-associated radioactivity is then measured in lysates using a gamma radiation counter and expressed with respect to protein content. By this method, differences in the level of any antigen retained by fixation can be easily quantified. The convenience, dynamic range of linearity and reproducibility of this technique compare favorably with Western blotting. Originally, the assay was designed to monitor the relative abundance of glial or neuronal cells in embryonic cerebral co-cultures upon various experimental conditions, by measuring related changes in glial fibrillary acidic protein (GFAP) or microtubule-associated protein 2 (MAP-2) content. It is proposed as a method of choice to quantify the effects of culture conditions or toxic agents on a specific cell type in mixed populations.
Nassogne
M C
MC
Cell Biology Unit, Christian de Duve Institute of Cellular Pathology, Brussels, Belgium.
Baudhuin
P
P
Courtoy
P J
PJ
D000818:Animals; D000941:Antigens / Q000032:analysis; D015415:Biomarkers; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis*; D015592:Immunoradiometric Assay / Q000379:methods* / Q000592:standards; D051379:Mice; D008815:Mice, Inbred Strains; D008869:Microtubule-Associated Proteins / Q000032:analysis*; D009457:Neuroglia / Q000737:chemistry*; D009474:Neurons / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000032:analysis
D000941:Antigens; D015415:Biomarkers; D005904:Glial Fibrillary Acidic Protein; D008869:Microtubule-Associated Proteins; D013205:Staphylococcal Protein A