Characterization of Murine Lymphocyte Subsets

IELs and LPLs were passed through a 40-um cell strainer to a obtain single-cell suspension. A single-cell suspension was stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) and incubated with brefeldin A (5 ug/ml) for 5 h, followed by staining for intracellular cytokines and surface markers. Exclusion of dead cells was performed with LIVE/DEAD Fixable Zombie Dead Cell Stain Kit (BioLegend). All cell preparations were Fc-blocked by CD16/32 antibody (BioLegend) prior to staining. Cell surface staining was performed with PerCP/Cy5.5 anti-mouse CD8α, FITC anti-mouse CD3, BV510 anti-mouse CD4, APC anti-mouse CD103, PE anti-mouse CD69 antibody and BV421 anti-mouse CD62L antibody (all from BioLegend). For detection of intracellular cytokines, cells were fixed in 4% PFA and permeabilized with BD perm/wash™ (BD Biosciences), followed by staining with Bv421 anti-mouse TNF-αand AF647 anti- mouse IFN-γ (BioLegend). Flow cytometric analysis was performed on an LSR Fortessa, and cell results were acquired using Diva software (BD Biosciences) and analyzed with FlowJo software. Sorting was performed on an Aria SORP high-speed cell sorter (BD Biosciences).