Anti cd138 281 2

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.

Single cell suspensions obtained from the intestinal mucosa or MLNs were stained and analyzed on FACSCanto II (Becton Dickinson, Franklin Lakes, NJ, USA) using FlowJo software v10.8.2 (TreeStar, Ashland, OR, USA). The following antibodies were used: anti-B220 (RA3-6B2; BioLegend), anti-CD38 (90; BioLegend), anti-CD45 (30-F11; BioLegend), anti-GL7 (GL7; BioLegend), anti-CD138 (281-2; BioLegend), anti-CD4 (GK1.5; BioLegend), anti-CXCR5 (L138D7, BioLegend), anti-PD-1 (29F.1A12, BioLegend), anti-Siglec-F (E50-2440, BD Biosciences, Franklin Lakes, NJ, USA), anti-CD11c (N418, BioLegend), anti-I-A/I-E (M5/114.15.2, BioLegend), anti-IgE (RME-1, BioLegend), anti-CD103 (W19396D, BioLegend), anti-CD11b (M1/70, BioLegend), anti-IL-7Rα (A7R34, BioLegend), anti-CCR6 (29-2L17, BioLegend), anti-TCRβ (H57-597, BioLegend), and anti-TCRγδ (UC7-13D5, BioLegend). Dead cells were gated out using a Zombie NIR Fixable Viability Kit (BioLegend). Before staining, Fc receptors were blocked with an anti-CD16/32 antibody (2.4G2, BioLegend). Negative controls consisted of isotype-matched, directly conjugated, nonspecific antibodies (BD Biosciences). Intracellular staining was performed using anti-Foxp3 antibody (FJK-16s; Thermo Fisher Scientific) and anti-RORγt antibody (Q31-378; BD Biosciences), as described previously [29 (link)].