Comprehensive T-cell Population Analysis by Flow Cytometry

T-cell populations were analyzed by flow cytometry using procedures previously described^{46 (link)}. Briefly, MLNs were harvested, homogenized, and resuspended in PBS containing 2% fetal bovine serum. For all samples, Fc receptor blocking was performed with Purified Rat Anti-Mouse CD16/CD32 (BD Biosciences, San Jose, CA, USA). Cells were stained using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) to assess viability. anti-mouse CD45 (BioLegend, San Diego, CA, USA), anti-mouse TCRβ (BioLegend), and anti-mouse CD4 (BioLegend) antibodies were used for surface staining. A FOXP3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) was used to fix and permeabilize cells. Anti-mouse/rat Foxp3 (Thermo Fisher Scientific), anti-mouse/human T-bet (Thermo Fisher Scientific), and anti-Mouse RORγt (Thermo Fisher Scientific) antibodies were used for intranuclear staining. Mouse IgG1 Kappa Isotype Control (Thermo Fisher Scientific), Rat IgG1 Isotype Control (Thermo Fisher Scientific), and Rat IgG2a Kappa Isotype Control (Thermo Fisher Scientific) were used as isotype controls. Samples were analyzed with CytoFLEX (Beckman Coulter, Brea, CA, USA) and FlowJo version 10.8 (FLOWJO, Ashland, OR, USA).