Flow cytometry fixation and permeabilization buffer kit 1

Collagenase (Type VIII), dimethyl sulfoxide (DMSO), E-Toxate™ reagent from Limulus Polyphemus, fetal bovine serum (FBS), hyaluronidase, monosodium urate crystals (MSU), and RPMI-1640 cell medium were purchased from Sigma‐Aldrich Co. (Milan, Italy). Flow cytometry fixation and permeabilization buffer kit I, proteome profiler mouse cytokine array kit and recombinant mouse Gal-9 were purchased from R&D System (Milan, Italy). FACS buffer and conjugated antibodies from BioLegend (London, UK). Ficoll-Paque Plus (endotoxin tested, ≤ 0.12 EU/ml) was obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Unless otherwise stated, all the other reagents were from BioCell (Milan, Italy).

Hepatic non-parenchymal cells were stimulated with 20 ng/ml PMA and 1µg/ml Ionomycin in the presence of 10µg/ml Brefeldin A in RPMI 1640 medium (supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units ml^-1 penicillin and 100 mg ml^-1 streptomycin) at 37°C in a humidified incubator with 5% CO2.
Following 4h of stimulation, cells were washed and stained for the T cells surface markers TCRβ, CD3, CD4 and CD8. Then, cells were washed and fixed with the Flow Cytometry Fixation and Permeabilization Buffer Kit I (R&D Systems) for 30 min in 4°C. Cells were washed with permeabilization buffer and stained for intracellular IL-17A.