Fluorescein isothiocyanate anti cd45ra

PBMCs were stained with Horizon V500 anti-CD3, allophycocyanin-H7 anti-CD4, Horizon V450 anti-CD8, fluorescein isothiocyanate anti-CD45RA (BD Pharmingen, San Diego, CA, USA), phycoerythrin anti-CD25, peridinin-chlorophyll protein-Cy5.5 anti-CCR7 (BioLegend, San Diego, CA, USA), phycoerythrin-Cy7 anti-CD127 (eBioscience, San Diego, CA, USA), and allophycocyanin anti-CD31 (Miltenyi Biotec, Bergisch Gladbach, Germany) mAbs. PBMCs were first gated for CD3 expression, then for CD4 and CD8 markers, and finally for the expression of CD45RA and CCR7 to identify naïve CD4+/CD8+(CD45RA+CCR7+), central memory CD4+/CD8+ (T_CM; CD45RA-CCR7+), effector memory CD4+/CD8+ (T_EM; CD45RA-CCR7-), and terminally differentiated effector memory (TEM_RA;CD45RA+CCR7-) cells. T regulatory cells (Treg) were identified as CD4+CD25int/highCD127low/- lymphocytes. Treg were further phenotyped as CD4+CD25int/highCD127low/-CD45RA+CCR7+ naïve Treg, CD4+CD25int/highCD127low/-CD45RA-CCR7+ Treg_CM and CD4+CD25int/highCD127low/-CD45RA-CCR7-Treg_EM subsets. Recent thymic emigrants (RTE) were recognized as naïve CD4+ lymphocytes expressing the CD31 molecule.
Absolute count and percentage were calculated for each T and B cell subset. Data were acquired using an eight-colour FACSCanto II cytometer and analysed with FACS Diva software (BD Biosciences, San Jose, CA, USA).

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).