The oil-microcentrifuge assay: a rapid and sensitive method for analyzing specific [3H]estradiol binding in cultured...

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.