Permeabilization buffer

Cell pellets were resuspended in 100 μl of 1% fetal bovine serum (FBS; VWR) and incubated with either trypan blue (Invitrogen) or LIVE/DEAD Fixable Yellow stain (Invitrogen) for 5 min at room temperature and 30 min at 37°C, respectively. Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS. trypan blue cell viability was measured using Countess II Automated Cell Counter (Thermo Fisher Scientific). For flow cytometry, samples were fixed in fixation buffer for 30 min at 4°C, washed with permeabilization buffer (R&D Systems), centrifuged, and incubated with fluorescent antibodies (table S2) in permeabilization buffer at 4°C for 1 hour. Samples were then washed with 10% FBS in PBS, centrifuged, and resuspended in 10% FBS in PBS. Appropriate negative internal controls were used. Data acquisition was done with a NovoCyte 3000 (ACEA Biosciences) and data analysis with NovoExpress.

For T-bet and GATA-3 staining, SL were fixed with 500μl of fixation buffer (R&D systems, USA) and incubated for 10 min at room temperature. Cells were centrifuged at 2000rpm for 5 minutes and washed twice with PBS. Then, SL were resuspended with 150 μl of permeabilization buffer (R&D systems, USA). Cells were incubated with FITC conjugated anti-human monoclonal antibody T-bet (R&D Systems, USA) and with PE conjugated anti-human monoclonal GATA3 (R&D Systems, USA), and control isotypes (R&D Systems, USA). According to a previous study that assessed the similarities between T-bet and GATA-3 protein sequences of Homo sapiens and Canis lupus familiaris using BLAST (basic local alignment search tool) algorithm of the National Center for Biotechnology Information (NCBI), human T-bet and GATA-3 showed, respectively, 93% and 97% homology with the canine protein. Acquisition of 10,000 events were counted by experimental replicate on channel FL1 and FL2, and cytometric analysis was performed with an Accuri C5 Flow Cytometer (BD Biosciences, USA) using BD Accuri C6 software, version 1.0.264.21 (BD Biosciences, CA, USA).
Lymphocytes were gated by forward and side scatter. Mean fluorescence obtained on T-bet positive cells was divided by the mean fluorescence of positive GATA-3 cells, generating a T-bet (Th1)/GATA-3 (Th2) ratio for each treatment.

Antibodies employed in flow cytometric analysis were obtained from various commercial sources: anti-CD1d (Biolegend, 1B1), anti-αGalcer:CD1d (eBioscience, L363), CD86 (BD Bioscience, GL1), TCRαβ (eBioscience,IP26), F4/80 (eBioscience, BM8), IL17-A (Biolegend,TC11-18H10.1), NK1.1 (Biolegend, PK136), and CD3 (Biolegend, 17A2). Cells were blocked with anti-CD16/32 antibody (Biolegend) for 15 min before incubation with fluorescently labeled antibodies at a concentration of 2 μg/ml for 45 min on ice. Stained cells were washed once with FACS buffer and analyzed by FACSan (Becton Dickinson). For internal staining, cells stained with surface makers were fixed in 2% PFA Fixation buffer (eBioscience) at 4°C overnight, followed by 3 washes with permeabilization buffer (R&D) and incubation with permeabilization buffer for 20 min on ice before staining with internal antibody. Stained cells were washed once with permeabilization buffer and suspended in PBS for analysis. Flow cytometry data were processed with FlowJo software (Tree Star, inc.,).