Macrophage Cytokine Modulation by GPR55

Control THP-1 and foam cells, primed overnight with LPS and untreated or incubated with GPR55 agonist or antagonist (alone or in combination), were washed 3 times with PBS, collected by means of accutase and then stained with FITC-conjugated anti-CD36 specific antibody. Intracellular staining for tumor necrosis factor α (TNF-α) and IL-10 was performed prior to cytokine inhibition, by adding 1 μg/ml brefeldine A (Sigma) 4h before cell collection. Cells were then fixed with 4% formaldehyde (Sigma) for 10 min at room temperature, and were stained in saponine 0.5% with anti-TNF-α APC and anti-IL10 PE. Flow cytometry as also used to assess GPR55 expression by staining macrophages and foam cells with GPR55 antibody both at cell surface or intracellulary upon cell permeabilization with saponine 4% formaldehyde and 0.5% saponine. Flow cytometry analysis was performed on a FACSCanto Flow Cytometer (Beckton Dickinson, NJ, USA), as reported [31 (link)].