Intracellular flow cytometry

Lymph node cells were stimulated with 2 µg/ml H56 together with anti-CD28 (37.51; BD Biosciences) and anti-CD49d (9C10; BD Biosciences) for 1 h. Subsequently, 10 µg/well brefeldin A (Sigma-Aldrich, St. Louis, MO) and 0.7 µl/well monensin/GolgiStop (BD Biosciences Franklin Lakes, NJ) were added and cells were incubated for 5 h at 37 °C. After overnight storage at 4 °C, cells were washed in FACS buffer (1% FCS (VWR-Bie & Berntsen, Herlev, Denmark), 0.1% sodium azide (VWR, Radnor, PA) in PBS (Life Technologies, Carlsbad, CA), and stained for surface markers with 1 µg/ml anti-CD4-APC-eFlour780 (clone GK1.5) and 1 µg/ml anti-CD44-FITC (clone IM7) (both eBiosciences, San Diego, CA) for 30 min at 4 °C. Cells were washed with FACS buffer, before fixation and permeabilization using Cytofix/Cytoperm kit (BD Biosciences Franklin Lakes, NJ). Subsequently, cells were stained for intracellular cytokines with 1 µg/ml anti-IFN-γ PE-Cy7 (XMG1.2; eBiosciences, San Diego, CA), 1 µg/ml anti-TNF-PE (MP6-XT22; eBiosciences, San Diego, CA) and 1 µg/ml IL-17A for 20 min. Finally, cells were washed, re-suspended in FACS buffer, and analyzed using a FACSCanto flow cytometer (BD Biosciences Franklin Lakes, NJ) and FlowJo software version 10.