Measurement of estrogen receptors in intact cells by flow cytometry

BACKGROUND: Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. Materials and Methods ER-positive human breast cancer cells MCF-7 and T47D, and ER-negative MDA-MBA-321 cells, were fixed and permeabilized by three different protocols. The cells were then stained by indirect immunofluorescence, using two commercial antibodies to ER (MA1-310 and DAKO 1D5), or by direct immunofluorescence using FITC-labeled anti-idiotypic antibody clone 1D(5). The stained cells were analyzed by flow cytometry. RESULTS: The fixation of cells with a mixture of 0.25% paraformaldehyde and 70% methanol, permeabilization with 0.05% Triton X-100, and increasing antibody and antigen reaction time led to 80-99% of cells being stained with anti-ER antibodies. The relative brightness of ER immunostaining was as follows: anti-idiotypic antibody ID5 > MA1-310 > DAKO 1D5. CONCLUSIONS: Direct immunofluorescence with the FITC-labeled anti-idiotypic antibody of permeabilized cells resulted in improved specific staining of the ER, as compared to indirect immunofluorescence with anti-ER antibodies of fixed and permeabilized cells. Increasing the length of staining, and treatment of cells with Triton X-100, are both necessary to improve the staining of intracellular antigen for flow cytometric analysis.