Pge2 parameter assay kit

Human chondrocytes (HC) derived from normal human femoral cartilage were obtained from Cell Applications, Inc. (Merck KGaA). The cells were maintained in chondrocyte growth medium which was purchased from Cell Applications, Inc. (Merck KGaA) at 37°C in a humidified atmosphere containing 5% CO₂. For treatment with interleukin (IL)-1β (PeproTech, Inc., Rocky Hill, NJ, USA), the cells were seeded at 1×10⁴ cells/well in a 24-well plate. Following overnight incubation at 37°C in a humidified atmosphere containing 5% CO₂, the growth medium was changed and cells were stimulated with 1 ng/ml IL-1β in the presence of 10 or 100 µg/ml of EADE prior to further incubation at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. Supernatants were subsequently collected to measure the levels of PGE₂ using a PGE₂ Parameter Assay kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol.

Cytokine levels in culture supernatants from BMDMs or mouse serum was measured by enzyme-linked immunosorbent assay (ELISA) using capture and detection antibodies against IL-6, IL-1β and TNF-α (eBioscience) according to manufacturer’s protocols. PGE2 in culture supernatants was measured by PGE2 Parameter Assay Kit (R&D Systems) according to the manufacturer’s instructions. Culture supernatants of BMDMs stimulated as described above were harvested after 3 h for TNF-α and IL-1β, 6 h for IL-6, or 24 h for PGE2. For intracellular cytokines in BMDMs, cells were stimulated as described for 1 h before adding GolgiPlug and incubated for another 3 h to accumulate intracellular cytokines. The cells were fixed and permeabilized in BD Cytofix/Cytoperm solution and stained with fluorochrome-conjugated antibodies against IL-1β, IL-6, IL-12β, iNOS and TNF-α. Intracellular cytokine production were analyzed by flow cytometry performed on Attune Flow Cytometer (ThermoFisher Scientific); analysis was performed with FlowJo (BD Biosciences).