Anti erα clone c 542

Surface and intracellular phenotyping of T cells was performed by flow cytometry as previously described [9 (link), 18 (link)]. Allophycocyanin-conjugated anti-CD3, allophycocyanin- or phycoerythrin (PE)-conjugated anti-CD4, peridinin chlorophyll protein-conjugated anti-CD8, fluorescein isothiocyanate (FITC)-conjugated anti-CD95, PE-conjugated anti-CD25, PE-conjugated anti-HLA-DR, FITC-conjugated anti-IFN-γ, PE-conjugated anti-IL-4 (BD Biosciences, San Jose, CA), FITC-conjugated anti-IL-17A (eBioscience, San Diego, CA), anti-ERα (clone C-542, Abcam, Cambridge, UK), and anti-ERβ (clone 1531, Santa Cruz Biotechnology, Santa Cruz, CA) monoclonal (m)Abs were used. Anti-ER Abs were visualized by FITC-conjugated F(ab’)2 fragment secondary Ab (Abcam). Equal amount of mouse IgG isotype controls were run in parallel. To determine the frequency of T cell subsets, total lymphocytes were first gated by forward and side scatter and then additionally gated for CD4 or CD8 molecule expression. Acquisition was performed on a FACSCalibur flow cytometer (BD Biosciences) and 50,000 events per sample were run. Data were analyzed using the Cell Quest Pro software (BD Biosciences).

Immunofluorescence analysis was performed as previously described [9 (link)]. Briefly, purified T lymphocytes were fixed with 4 % formaldehyde and permeabilized with 0.5 % Triton X-100 in PBS. Cells were stained with anti-ERα (clone C542, Abcam) or anti-ERβ mAbs (clone 1531, Santa Cruz Biotechnology) and then incubated with Alexa Fluor 488-coniugated secondary antibody (Molecular Probes, Eugene, OR). Nuclei were counterstained with Hoechst 33342 (Molecular Probes). Fluorescence was analyzed with an Olympus U RFL microscope (Olympus, Hamburg, Germany).