Fluorescein isothiocyanate anti igd

One million PBMCs were phenotyped after staining with peridin-clorophyll protein-Cy5.5 anti-CD19, phycoerythrin Cy7 anti-CD10, fluorescein isothiocyanate anti-IgD, and phycoerythrin anti-CD27 (BD Pharmingen, San Diego, CA, USA) monoclonal antibodies (mAbs). Cells were initially gated for CD19 expression, and then for CD10 marker to identify immature CD19+CD10+ B cells and mature CD19+CD10- B cells. Mature B cells were examined for IgD and CD27 molecule expression to recognize naïve IgD+CD27- B cells, unswitched memory IgD+CD27+ B cells, switched memory B cells (IgD-CD27+), and double negative B cells (DN; IgD-CD27-).

TRECs and KRECs were measured, as previously described^{52 (link)}, by duplex real-time PCR (7500 Fast real-time PCR system; Applied Biosystems Foster City, CA) in DNA extracted from peripheral blood mononuclear cells with QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA). Both were expressed per ml of blood because their number was corrected for lymphocyte plus monocyte count in 1 ml of blood.
T- and B-cell subsets were determined by cytofluorimetric analysis (BD FACSCanto II, BD Biosciences, San Jose, CA). Briefly, phycoerythrin anti-CD3, allophycocyanin-H7 anti-CD4, phycoerythrin-Cy7 anti-CD8, fluorescein isothiocyanate anti-CD45RA (BD Pharmingen, Heidelberg, Germany), peridinin-chlorophyll protein-Cy5.5 anti-CCR7 (BioLegend, San Diego, CA) monoclonal antibodies were used for T-cell subsets characterization. For B-cell subpopulation identification, peridinin-chlorophyll protein-Cy5.5 anti-CD19, phycoerythrin-Cy7 anti-CD10, fluorescein isothiocyanate anti-IgD, and phycoerythrin anti-CD27 monoclonal antibodies (BD Biosciences) were used. Data were analyzed using FACS Diva software (BD Biosciences).