Flow cytometric determination of estrogen receptors in intact cells

Flow cytometry has been used to detect estrogen receptor (ER) in intact cells, using 1-(N)- fluoresceinyl estrone thiosemicarbazone ( 17FE ), a ligand shown to have sufficient affinity and specificity to identify high-affinity receptors. For each concentration of 17FE , two fluorescent distributions were obtained: (a) "total" fluorescence due to 17FE alone; and (b) diethylstilbestrol-inhibited fluorescence ("nonspecific"). The mean fluorescence intensity for both the "total" (MT) and "nonspecific" (MN) distributions was calculated at each ligand concentration by obtaining the total brightness of each sample, normalized to the number of cells analyzed. A specific binding curve was constructed over a broad range of 17FE concentrations (1.5 X 10(-9) to7 .5 X 10(-7) M) by plotting the mean specific fluorescence intensity (MS = MT-MN) at each 17FE concentration. Saturable binding was demonstrated for a known ER-positive cell line (MCF-7) at low ligand concentrations (5 to 10 X 10(-8) M), while no specific binding was obtained for a known ER-negative cell line (MDA-231). The Kd was estimated from the specific binding curve of MCF-7 cells as the concentration of 17FE at half-maximal binding. When corrected for the binding activity relative to estradiol, a Kd of 18 X 10(-10) M was obtained. The flow cytometer-generated distributions give a qualitative estimate of the proportions of ER-rich and ER-poor cells. Quantitation of ER heterogeneity will require a mathematical algorithm expressing the difference between the "total" and "nonspecific" distributions. These studies demonstrate that flow cytometry and an appropriate ligand can detect and partially describe ER heterogeneity in intact cells.