Allophycocyanin conjugated anti cd3

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).

Flow cytometry was performed using a FACS Canto II (BD Biosciences, Mountain View, CA) and the results were analyzed using FlowJo software (Tree Star Software, San Carlos, CA). The PBMCs were stained immediately after purification with the following monoclonal antibodies: fluorescein isothiocyanate-conjugated anti-CD56, allophycocyanin-conjugated anti-CD3, phycoerythrin-conjugated anti-CD 69 (all from BD Pharmingen, San Diego, CA). The PBMCs were then incubated with antibodies for 30 minutes at 4°C in the dark. Dead cells were excluded from the analysis using 7-AAD (7-amino-actinomycin D, BD Pharmingen) or propidium iodide staining. Because of limited cell numbers, all surface markers could not be measured in every sample. In this analysis, T cells were defined as CD56^- CD3⁺ cells, NK cells as CD56⁺ CD3^- cells, and NKT cells as CD56⁺ CD3⁺cells (for the gating strategy see Figure S1, Supplemental Digital Content, http://links.lww.com/MD/J704).^{[16 (link)]}