Immunophenotyping of Th17 and Treg Cells

Single-cell suspensions of spleen were generated as has been described previously (Taylor et al., 2018 (link)). Blood was obtained from SS and SR rats, which had taken 20% fructose or tap water for 4 weeks. PBMCs were separated by Ficoll-Paque PLUS gradient centrifugation (GE Healthcare, Chicago, IL, USA). Briefly, phenotypic and intracellular analyses were performed by incubating cells with antibodies for T cell surface markers – fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat CD3 (1:100; clone G4.18, #554832; BD Biosciences, Franklin Lakes, NJ, USA) and PE-conjugated mouse anti-rat CD4 (1:100; clone OX-35, #554838; BD Biosciences) – for 30 min on ice in the dark. After washing, the cells were fixed and permeabilized using fix/perm concentrate (BD Biosciences) before incubation with antibodies for intracellular staining of APC rat anti-rat IL-17A (1:100; clone eBio17B7, #17-7177-81; Thermo Fisher Scientific) to identify Th17 cells and PerCP-cyanine5.5 rat anti-rat FOXP3 (1:100; clone FJK-16s, #14-5773-82; Thermo Fisher Scientific) to identify Treg cells. Cells were then washed and run through a four-color flow cytometer (FACSCalibur; BD Biosciences) and data were collected using CellQuest or FlowJo v10.0 software. An example of the flow cytometry gating strategy used is shown in Fig. S3.