Pe igg2a κ

Cells were surface-stained and analyzed by flow cytometry on LSRII flow cytometers (Becton Dickinson) using Cy5-, APC- or FITC-anti-IgM (μ-chain-specific, Southern Biotech), PE-anti-λ5 (LM34, a gift from A. G. Rolink), PE-Cy7-B220 (eBioscience), PE-B220 (BD Pharmingen) APC-CD19 (eBioscience), PE-CD43 (BD Pharmingen), PE-κ (Southern Biotech), PE-CD11b (eBioscience) and APC-CD127 (eBioscience). Intracellular stainings were performed using Fix and Perm cell permeabilization Kit (ADG) according to the manufacturer’s instructions. Antibodies used for intracellular FACS-staining were PE-anti-Pax5 (eBioscience), anti-SLP-65 (monoclonal IgG mouse, self-produced), anti-AKT (Cell Signaling), anti-FoxO1 (Cell Signaling), pAKT (Cell Signaling), APC-anti-rabbit IgG1 (AbD Serotec) and APC-anti-mouse IgG (AbD Serotec). PE-IgG2a κ (eBioscience), APC-IgG1 (eBioscience), APC-IgG2a κ (eBioscience) were used as isotype controls. PE-anti-Biotin (BioLegend), Alexa Fluor 647-anti-mouse-IgG Fab2 and Alexa Fluor 647-anti-rabbit-IgG Fab2 were used as secondary antibodies or as staining controls, respectively when used without the primary antibody.

Splenic and MLN mononuclear cells were isolated by lympholyte-M enrichment (Cedarlane, Burlington, NC, USA) as described [50 (link)]. Colonic tissues were digested using Type IV collagenase (Sigma Aldrich) as described elsewhere [51 ] and lymphocytes were enriched over 70/45% Percoll gradient (Sigma Aldrich). Surface and intracellular staining were performed as reported previously [45 , 52 (link)]. Cells were surface-stained with APC-anti-CD4 (clone L3T4, eBioscience, San Diego, CA, USA) followed by intracellular detection of PE-anti-FOXP3 (clone FJK-16s), PE-anti-IL-17A (clone eBio17B7), PE-anti-IFNγ (clone XMG1.2), or PE-anti-IL-4 (clone 11B11) (eBioscience). Isotype controls utilized were PE-IgG2_aκ, PE-IgG1κ, and APC-IgG2_bκ (eBioscience). Flow cytometric analysis was conducted using a BD Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA).