Anti cd19 1d3

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.

DCs from spleens of BALB/c mice were prepared using the GentleMACS dissociator (Miltenyi Biotech) according to the manufacturer's protocol. Briefly, spleens were dissociated in GentleMACS C tubes in medium containing collagenase and DNase, incubated for 15 min at 37 C before adding EDTA at a final concentration of 10 mM. Erythrocytes were lysed by incubation with ACT buffer for 5 min on ice. Finally, cells were filtered through a 75 mm Nylon cell strainer. The following Abs were used for subsequent flow cytometry analysis: anti-CD3e (145-2C11; Tonbo Bio- sciences), anti-CD19 (1D3; Tonbo Biosciences), anti-CD49b (DX5; eBioscience), anti-Ly6G (1A8), anti-CD45R (RA3-6B2; Tonbo Biosciences), anti-MHC-II (M5/114.15.2; BioLegend), anti-CD11c (N418; Tonbo Bio- sciences), anti-CD11b (M1/70; Tonbo Biosciences), and anti-CD24 (M1/69; BioLegend).

The following rat anti-mouse mAbs; anti-CD11b (M1/70, TONBO Biosciences) and anti-CD19 (1D3, TONBO Biosciences), and mouse or rabbit anti-human mAbs; anti-CD38 (HB7, TONBO Biosciences), anti-p21 (12D1, Cell Signaling Technology), anti-AIM2 (3B10, BioLegend), anti-SKP2 [EPR3305(2), Abcam], and anti-USP10 (D7A5, Cell Signaling Technology) were used. Isotype-matched control IgGs for individual rat and mouse mAb were purchased from BD Biosciences. PE-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific) was used as the secondary Ab for the flow cytometry analysis.