Dynamic Mass Redistribution Assays
Untransfected CHO or CHO cells expressing MRGPRD were plated in Corning® Epic® 384 well microplates at 10,000 cells.well-1 in 30µl media for 30 minutes at room temperature before incubating overnight at 37°C with 5% CO2. On the day of experimentation, cells were washed with fresh media without FBS and incubated in the microplates for 2 hours at room temperature before the DMR baseline was measured. Cells were treated with antagonist (Thioridazine Hydrochloride, PD123,319 and MU-6840) or vehicle for 30 minutes prior to the addition of agonist. DMR measurements were made for 60 minutes using a PerkinElmer EnSpire plate reader. All antagonists were solubilised in DMSO and diluted in assay buffer comprising HBSS plus 20mM HEPES and 0.01% Pluronic F-127 (resulting in a final 0.1% DMSO concentration). β-alanine, GABA and glycine were solubilised and diluted in assay buffer. When studying the effects of signalling pathway modulators, the Gαi/oinhibitor Pertussis toxin (PTX) was used at 0.1µg.ml-1and incubated overnight while the Gαq inhibitor, YM-254890 was used at 10µM and pre-incubated with cells for 30 minutes. All data are expressed as percent effect relative to the maximum β-alanine response and the unstimulated controls at 4 minutes using internal plate controls to normalise for plate-to-plate and day-to-day variability.