Calcium imaging experiments with HEK-293 (ATCC) overexpressing GCaMP6s (an ultrasensitive fluorescent protein calcium sensor) were used to assay P2Y1 activation. HEK-GCaMP6s cells were grown in DMEM:F12 (Invitrogen) containing 5% FBS, 0.3 mg/ml G418, and 1× penicillin/streptomycin (Invitrogen). For calcium imaging experiments, HEK-GCaMP6s cells were subcultured into 1% gelatin-coated 96-well cell culture plates and grown to 80-90% confluence. Before imaging, the medium was replaced with LHC-9 medium (Invitrogen; Carlsbad, CA) containing 0.75 mM trypan red and, for antagonist treatment wells, the P2Y1 antagonist MRS2179 or molleamine C, and incubated for 30 min before assessing P2Y1 activation. The agonist MRS2365, used to activate P2Y1, was prepared in LHC-9 at 3× concentration and added to cells at 37 °C as previously described.59 (link), 60 (link) Calcium flux was detected using a NOVOstar fluorescent plate reader (BMG LABTECH).