The oil-microcentrifuge assay: a rapid and sensitive method for analyzing specific [3H]estradiol binding in cultured cells.
We have developed an oil- microcentrifuge assay system for analyzing the binding of [3H]estradiol in metabolically active MCF-7 and MDA human breast cancer cells. Complete separation of 2 X 10(6) cells from radioactive media can be achieved within 5 s of centrifugation at 12,000 rpm. The [3H]estradiol binding sites in MCF-7 cells are filled within 20 min of radioligand exposure. Using this assay, our MCF-7 cells contain approximately 15,000 high affinity and saturable binding sites. Binding is inhibited by estradiol and tamoxifen but not progesterone. There is no specific binding of [3H]estradiol in MDA cells. This assay is a rapid, sensitive and reproducible method for investigating hormone-receptor binding and ligand specificity in cultured cells; results compare favorably with those obtained by more complex and lengthy techniques.
D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000032:analysis; D002460:Cell Line; D002469:Cell Separation; D002478:Cells, Cultured; D002498:Centrifugation; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D009821:Oils; D011960:Receptors, Estrogen / Q000378:metabolism*; D013997:Time Factors
D009821:Oils; D011960:Receptors, Estrogen; D004958:Estradiol