Novoexpress

The cell surface markers and cytokines were analyzed by flow cytometry, as described previously [30 (link),31 (link),32 (link)]. Living cells were stained with the following antibodies in PBS containing 0.1% (weight/volume) bovine serum albumin and 0.1% NaN₃ for 30 min on ice. The following antibodies were obtained from BD Biosciences (Lake Franklin, NJ, USA): anti-CD4 (GK1.5), anti-B220 (RA36B2), anti-CXCR5 (2G8), anti-T- and B-cell activation antigen (GL-7), anti-CD95 (Jo2), anti-CD138 (281-2), and anti-IgD (11-26c.2a). The following antibodies were obtained from eBioscience (Thermo Fisher): anti-PD-1 (J43), anti-CXCR5 (SPRCL5), and anti-IL-21 (FFA21). To detect cytokine secretion, cells were stimulated with phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich) and ionomycin (PeproTech-BioGems, NJ, USA) for 5 hours. The cells were fixed using a Fixation/Permeabilization Solution Kit (BD Biosciences). To detect the metabolism-related regulators, we used anti-Glut1 (EPR3915) and anti-SDHα (EPR9043B) antibodies. Cells were fixed with a Fixation/Permeabilization Solution Kit (BD Biosciences) and were intracellularly stained for both molecules. All flow cytometry data were obtained with ACEA NovoCyte (ACEA Biosciences, Inc., San Diego, CA, USA), and the data were analyzed with NovoExpress (TreeStar, San Carlos, CA).

Colon tissue samples were isolated, rinsed three times using cold PBS, and digested for 45 min at 37 °C in DMEM containing 2% FBS, collagenase IV (1 mg/mL; Sigma-Aldrich), and DNase I (10 U/mL; Roche). The resultant digest was then passed through a 70-μm mesh filter to generate a single-cell suspension, which was stained with LIVE/DEAD, anti-CD45, anti-CD11b, anti-F4/80, anti-CD4, anti-CD8, and anti-B220 prior to analysis using a BD LSR Fortessa™ Flow Cytometer. All data were analyzed using NovoExpress or FlowJo X (TreeStar, CA, USA).