Cd45r b220 percp cy5

For surface staining of human specimens, cell suspensions were incubated with the appropriate combination of the following monoclonal antibodies: CD19-PC5 (J3-119, Beckman Coulter), CD80-Brilliant Violet 421™ (2D10, BioLegend^®), CD83-APC (HB15e, BioLegend^®), CD86-APC (IT2.2, BioLegend^®), and HLA-DR-V500 (G46-6, BD Horizon™). For surface staining of mouse specimens, cell suspensions were incubated with Fc-blocking antibody (Bioxcell, 2.4G2) to avoid unspecific Fc-Receptor binding. After washing, the cells were incubated with the appropriate combination of the following ­antibodies: CD11b-PECy7 (M1/70, BioLegend^®), Ly6G-PE (1A8, BD Biosciences), Ly6C-Biotin (AL-21, BD Biosciences), CD3-APC(17A2, BioLegend^®), CD45R-B220-PerCP-Cy5.5 (RA3-6B2, eBiosciences). To detect anti-Ly6C-Biotin antibody binding, cells were subsequently stained with streptavidin-FITC (Dako). The samples were acquired with BD LSRFortessa (BD Biosciences), and results were analyzed with FlowJo software (Tree Star, Inc.).

Cells from the inguinal lymph nodes were collected from all individuals; the cells were plated (10⁶ cells/well) and treated with Mouse BD Fc Block™ (BD Pharmingen, 553142) to block non-antigen-specific bindings of immunogloblulins to Fcγ III and Fcγ II receptors. Two panels of antibodies were used to stain cell populations in the germinal centers (GC) of the lymph nodes; GC B cells (B220⁺ IgD⁻ CD38⁻ GL7⁺) and T follicular helper cells (TFH) (B220⁻ CD4⁺ PD-1⁺ CXCR5⁺). The following antibodies were mixed in PBS 1% FBS: GL7-FITC (BioLegend GmbH, Koblenz, Germany; 144604), IgG1-PE (BD Pharmingen, 550083), B220-PerCP-Cy5.5 (BD Pharmingen, 552771), CD38-PE-CY7 (eBioscience, 25-0381) and IgD-BV786 (BD Pharmingen, 563618) to stain the B cells in the GC. To stain the TFH cell population, the following antibody panel was used: CD4-FITC (BD Pharmingen, 553047); CD279(PD1)-PE (BD Pharmingen, 551892); CD45R(B220)-PerCP-Cy5.5 (eBioscience, 65-0865-14), CXCR5-BV421 (BD Pharmingen, 562889) and live/dead-EF780 (BD Pharmingen, 562889). After an incubation period of 30 min at 4 °C, the cells were acquired on the FACS LSRII instrument using DIVA software (BD Bioscience, USA) and all the acquired data analyzed using FlowJo software (Tree Star Inc.). Compensation beads (BD Pharmingen, USA) were used to compensate the fluorophores.