Anti cd23 b3b4

Surface staining of cells was performed by suspending them in blocking solution (10% fetal calf serum in PBS) for 30 min at 4°C in the presence of the following fluorescent-labeled mAbs: anti-B220 (RA3-6B2, eBioscience), anti-CD3 (145-2C11, TONBO Bioscience), anti-CD4 (GK1.5, BioLegend), anti-CD8 (53-6.7, TONBO Bioscience), anti-CD25 (PC61.5, eBioscience), anti-CD21 (7G6, eBioscience), anti-CD23 (B3B4, eBioscience) and anti-LAP (TW7-16B4, BioLegend), anti-CXCR5 (SPRCL5, eBioscience), anti-PD1 (J43, eBioscience), anti-CD138 (281-2, BioLegend), anti-CD19 (1D3, TONBO Bioscience), anti-GL-7 (GL7, eBioscience), anti-IgM (eb121-15-F9, eBioscience), anti-IgD (11-26c, eBioscience), anti-CD40L (MR1, eBioscience), and anti-CD69 (H1.2F3, BioLegend). For intracellular cytokine staining, cells were exposed to monensin, fixed and permeabilized using the BD Cytofix/Cytoperm Plus Kit with BD GolgiStop™ (BD Biosciences) following manufacturer’s indications, and then stained with specific fluorescent-labeled mAbs: anti-IFN-γ (FITC-XMG1.2, TONBO Bioscience) and anti-IL-2 (PE-JES6-5H4, BD Pharmingen). For FoxP3 staining, the Anti-Mouse/Rat Foxp3 PE Staining Set (eBioscience) was used following manufacturer’s indications. Flow cytometry analyses were performed on a FACS Canto II equipped with CellQuest (BD Biosciences) and Flowjo 8.7 software.

Cells from the thymus and single-cell suspensions from the spleen or lymph nodes were stained with combinations of following antibodies: anti-mouse CD8α (53–6.7), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-TCRβ (H57-597), anti-CD11b (M1/70), and anti-Gr1 (RB6-8C5), from Tonbo Biosciences (San Diego, CA, USA); anti-CD4 (GK1.5) and anti-B220 (RA3-6B2) from BD Biosciences (Franklin Lakes, NJ, USA); and anti-TCRγδ (GL-3), anti-F4/80 (BM8), anti-CD21 (8D9), and anti-CD23 (B3B4) from eBiosciences (San Diego, CA, USA). All samples were resuspended in phosphate-buffered saline (PBS) staining buffer containing 2% fetal bovine serum and 0.01% NaN₃ and pre-incubated for 15 min at 4 °C with 2.4G2 supernatant to block the Fc receptor. Samples were then washed and stained with specific mAbs for 20 min at 4 °C. Data were collected on a FACSCanto II (BD Biosciences) and analyzed using FACS Diva (BD Biosciences) or FlowJo (Tree Star, OR, USA) software.