PBMC Co-culture with Stromal Cells

Stromal cells were seeded overnight, and the next day, PBMCs were added at a 5:1 ratio, with or without phytohemagglutinin (PHA, 5 μg/ml), as described [11 (link), 12 (link)]. Treatments, IL-23 (50 ng/ml), anti-IL-23 antibody (1 μg/ml) (R&D Systems, Minneapolis, USA), or control irrelevant antibody (1μg/ml) (Dendritics, Lyon, France) were added to cell cultures, cells alone or co-cultures. After 48 h, supernatants were collected for the analysis and cells for CD3, CD4, CD69, and CD86 staining.
For mRNA expression, co-cultures were initiated by seeding synoviocytes or skin fibroblasts overnight in 12-well plates at a density of 15 × 10⁴ cells/well in a complete RPMI 1640 medium (Eurobio, Courtaboeuf, France, RPMI medium supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 100U/ml penicillin/streptomycin). The next day, PBMCs (75 × 10⁴ cells/well) were seeded in a complete RPMI medium, without or with PHA (5 μg/ml) or IL-23 (50 ng/ml). After 24 h, cells were recovered for RNA extraction.