Cell experiment:

Bone marrow-derived macrophages treated with either vehicle (0.3% DMSO) or 1 μM 6-OAU for 1 h are incubated with unopsonised pHrodo E. coli bioparticles at 0.1 mg/mL in a 96-well flat clear bottom plate. For the inhibition studies with GPR84 antagonist 8, cells are pretreated with 10 μM GPR84 antagonist 8 for 30 min before addition of either vehicle or 6-OAU. The plate is then placed into the IncuCyte Zoom platform which is housed inside a humidified incubator at 37℃, 5% CO2. Two to four images per well from three technical replicates are taken every 15 min for 4 h[1].

GPR84 antagonist 8 is a selective GPR84 antagonist.

GPR84 is a member of the metabolic G protein-coupled receptor family, and its expression has been described predominantly in immune cells. To test the hypothesis that blocking the activation of GPR84 can be a potential anti-inflammatory strategy in different inflammatory diseases, GPR84 antagonist 8 is used. The potency and specificity of GPR84 antagonist 8 is assessed tusing GPR84-CHO cells in the cAMP assay. GPR84 antagonist 8 effectively inhibits the action of 6-OAU in decreasing cAMP production in GPR84-CHO cells. To test GPR84 antagonist 8's inhibition of the pro-inflammatory effects of GPR84 activation in macrophages, LPS pre-treated BMDMs are incubated with 10 µM GPR84 antagonist 8 for 30 min before adding 1 µM 6-OAU. Protein analysis by Western Blot shows that the GPR84 antagonist 8 partially blocks the phosphorylation of AKT and ERK induced by 6-OAU[1].

[1]. Recio C, et al. Activation of the Immune-Metabolic Receptor GPR84 Enhances Inflammation and Phagocytosis in Macrophages. Front Immunol. 2018 Jun 20;9:1419.