Anti cd19 pe

Blood was taken from the mice on day 56, stimulated, and analyzed for basophil activation as previously described by Torrerro et al.19 In summary, whole blood was diluted 1:1 using RPMI in heparinized tubes. After incubation with anti‐mouse IgE at 0.125 µg/mL (R35‐72, BD Biosciences) or PE at 20 µg/mL for 90 min at 37°C in 5% CO₂, activation was stopped with PBS containing EDTA in 12 × 75 mm test tubes. The Whole Blood Lysing Reagents was used to lyse the red blood cells and fix the samples according to protocol (Beckman Coulter, Fullerton, CA). To block the Fc‐receptor, cells were incubated with anti‐CD16/CD32 (clone 2.4G2) for 20 min. Cells were stained for 30 min at 4°C with the following fluorescent‐labeled antibodies: anti‐IgE‐FITC (1:100, clone 23G3), anti‐CD49b‐APC (1:200, clone CX5), anti‐CD4‐PE (1:200, clone RM4‐5), anti‐CD200R‐Percpefluor 710 (1:200, clone OX110), and anti‐CD19‐PE (1:200, clone 6D5) from eBioscience. Acquisition of the samples was performed on the BD Accuri™ C6 flow cytometer, analysis with BD sampler software (BD Biosciences). Fluoresence minus one technique was used to set the gates.

Heparinised whole blood was stained with the following directly conjugated monoclonal antibodies: anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-FITC, anti-CD8-phycoerythrin (PE) and anti-CD19-PE (eBioscience, San Diego, CA, USA). Following lysis, the cells were analysed using a BD FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). Results were expressed as the absolute number of cells.

Spleen tissues were obtained from mice at 15–20 weeks after primary immunization, and the B- and T-cell populations of interest were identified using the following specific antibodies (eBioscience): anti-B220-APC, anti-GL7-FITC (or PerCP), anti-CXCR5-PerCP, anti-CD138-PE, anti-ICOS-PE (or APC), anti-IgD-FITC, anti-IgM-PE, anti-CD19-PE (or PerCP), anti-CD5-FITC, anti-IL-10-FITC, anti-Foxp3-PE, anti-CD4-PerCP (or FITC, PE), anti-CD25-APC, anti-Foxp3-FITC (or APC), anti-IL-17-PE, anti-IL-4-PE (or FITC), and anti-IFN-γ-FITC (or PE). Stained sections were visualized by confocal microscopy (LSM 510 Meta, Carl Zeiss, Oberkochen, Germany). The expression of GC or plasma B cells were estimated by comparing the mean fluorescence intensity using LSM 510 Meta, Carl Zeiss software.

Ethical approval was obtained from the TCD School of Biochemistry and Immunology Research Ethics Committee for experiments involving PBMCs. Human peripheral blood mononuclear cells (PBMCs) from anonymous healthy donors were obtained by informed consent from buffy coats of blood packs from the Irish Blood Transfusion Service, using Lymphoprep (Axis-Shield) gradient centrifugation. For cell sorting to analyse constituent cells, PBMCs were stained with anti-CD14 APC, anti-CD19 PE, anti CD3 PE-Cy5.5 and anti-CD56 PE-Cy7 (eBiosciences). Cells were fixed and permeabilised using the FoxP3 staining buffer set (eBiosciences) and were stained with anti-MNDA FITC (Cell Signalling). Labelled cells were analysed on a FACSCanto II flow cytometer (BD Biosciences) and were evaluated with FlowJo software (TreeStar). Monocytes were isolated from PBMCs by positive selection using CD14 beads (Miltenyi Biotech), following the manufacturer’s instructions. Macrophages were generated by growing the monocytes in the presence of 50 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF, Sigma–Aldrich) or 20 ng/ml macrophage colony stimulating factor (M-CSF, Sigma–Aldrich), for 7 days, changing the media every 2–3 days.