title: Measurement of estrogen receptors in intact cells by flow cytometry.
abstract: 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.
authors.affiliation: Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, Texas 77555-1110, USA.
authors.forename: S
authors.initials: S
authors.lastname: Cao
authors.forename: S D
authors.initials: SD
authors.lastname: Hudnall
authors.forename: F
authors.initials: F
authors.lastname: Kohen
authors.forename: L J
authors.initials: LJ
authors.lastname: Lu
mesh_terms: D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001943:Breast Neoplasms / Q000276:immunology / Q000378:metabolism*; D002463:Cell Membrane Permeability; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D019085:Fluorescent Antibody Technique, Direct; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D017830:Octoxynol / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000032:analysis* / Q000276:immunology; D013501:Surface-Active Agents / Q000494:pharmacology; D016707:Tissue Fixation; D014407:Tumor Cells, Cultured
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D011960:Receptors, Estrogen; D013501:Surface-Active Agents; D017830:Octoxynol