Determination of cAMP in primary microglia

The brain tissue from GPR110-WT or KO mice at 4 months of age were dissociated into single cells using the Adult Brain Dissociation Kit (Miltenyi Biotec, Gaithersburg, MD, USA, cat#130-107-677) according to the manufacturer’s instruction. To isolate the microglia, the single-cell suspension was incubated with CD11b Microbeads for 30 min at 4 °C. The cells were resuspended in MACS buffer and passed through the LS column (Miltenyi Biotech). The column was washed 4 times with MACS buffer and then magnetically labeled CD11b-positive cells were flushed out of the columns twice using MACS buffer. Enriched CD11b-positive microglia cells were pelleted by centrifuging at 300 × g at 4 °C for 10 min and resuspended in DMEM media supplemented with 10% fetal bovine serum (FBS). Isolated CD11b-positive microglia cells were cultured in poly-D-lysin coated 384-well white/clear bottom plate for 7 days by changing 50% of DMEM media supplemented with 10% FBS every other day. On the day of the cAMP assay, media was changed to the stimulation media consisting of phenol red-free DMEM supplemented with 0.5 mM IBMX (Tocris, Minneapolis, MN, USA), a phosphodiesterase inhibitor. Microglia cells were treated with different concentrations of synaptamide or A8 complexed with 0.05% fatty acid-free BSA (Sigma-Aldrich) in the presence of 40 μM vitamin E for 10 min. Separately, microglia cells were treated with 10 μM Forskolin (Tocris, cas# 66575-29-9) for 10 min. The cAMP production was measured by a homogenous time-resolved fluorescence assay with a cAMP Gs Dynamic Kit (Cis Bio, Bedford, MA, USA, cat#62AM4PEB) using the FlexStaion 3 device (Molecular devices)