Cytokine and Chemokine Measurements

To assess the effects of OSM on pro-inflammatory mediators, RAFLS/HUVEC were seeded in 48-well plates at a density of 3 × 10⁵ and allowed to attach overnight. Cells were incubated in serum-free RPMI-1640 or MCDB-131 for 24 h and subsequently stimulated with OSM (10 ng/ml). For synergy experiments, cells were also incubated in the presence or absence of TNFα (0.01, 0.1, 1 ng/ml). Supernatants were harvested and levels of IL-6, MCP-1, IL-8, RANTES, and GROα were measured by specific ELISA (MCP-1: eBiosciences, USA, IL-6, IL-8, RANTES, GROα; R&D systems, UK) according to manufacturer's conditions.