Human il 1ra il 1f3

Enzyme-linked immunosorbent assay (ELISA) was used to determine the presence of IFNγ, TNFα, and IL1RN in the supernatant of macrophages treated with rat normal, metaplastic and neoplastic ECM after 6 or 24 hrs. Supernatants were first centrifuged at 1500 rpm at 4°C for 10min to remove any particulate material. The ELISA for IFNγ (Human IFNγ ELISA Kit II, BD OptEIA), TNFα (Human TNF ELISA Kit II, BD OptEIA), and IL1RN (Human IL-1ra/IL-1F3, R&D Systems) was performed according to manufacturer’s instructions at the appropriate dilution (n=5, technical duplicate). To evaluate the similarity between rat and human ECM treatment on macrophages, the supernatant of macrophages treated with human normal, metaplastic, and neoplastic ECM was similarly measured for TNFα concentration by ELISA (n=4, technical duplicates). The human ECM samples were either patient-matched (“n1”), distinctive (“n2”), or combined patient-matched (3 patients combined for “n3,” and 2 patients combined for “n4”).