Tongues were harvested and digested with Collagenase IV (0.7 mg/ml) in Hanks’ balanced salt solution for 30–45 mins at 37°C. Cells were separated by Percoll gradient centrifugation. Abs were from the following sources: anti-CD45 and anti-CD11b (BioLegend), anti-CD4 and anti-F4/80 (Invitrogen), anti-TCRβ, anti-CD8, and anti-CD19 (eBioscience), and anti-Ly6G and anti-Ly6C (BD Biosciences). Dead cells were excluded using Ghost Dye (eBioscience). Data were acquired with an LSRFortessa and analyzed using FlowJo software (TreeStar).
Collected single-cell suspensions from LN were filtered and dead cells were excluded using Ghost Dye (eBioscience). LN were cultured in complete medium (RPMI media containing 10% FCS, supplemented with L-glutamine and antibiotics) with 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of Golgiplug (BD Biosciences) for 4 h, followed by staining with Ghost Dye, CD4 and IL-17 (Biolegend).
Collected single-cell suspensions from LN were filtered and dead cells were excluded using Ghost Dye (eBioscience). LN were cultured in complete medium (RPMI media containing 10% FCS, supplemented with L-glutamine and antibiotics) with 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of Golgiplug (BD Biosciences) for 4 h, followed by staining with Ghost Dye, CD4 and IL-17 (Biolegend).