HEK-293 cells (ATCC CRL-1573.3) were cultured in growth media [DMEM (Gibco), 10% heat inactivated fetal bovine serum (Gibco), penicillin (100units/mL), streptomycin (100μg/mL) and L-glutamine (290μg/mL)] and used for assays between passages 5 and 25. For cestode GPCR heterologous expression assays, cells were transfected (Lipofectamine 2000, Invitrogen) at 80% confluency approximately 16 hours after seeding within T-25 culture flasks with a 1:1 ratio of human codon optimized cestode GPCR cDNA (subcloned into a pcDNA3.1(-) mammalian expression vector) and cDNA encoding the pGloSensor 22-F plasmid (Promega). The following day, cells were trypsinized, centrifuged (300g/5min), resuspended in DMEM supplemented with 1% dialyzed FBS (Gibco) and plated in 96 well, solid white plates (Corning, cat # 3917). After overnight culture to allow adherence, media was exchanged for assay buffer (HBSS supplemented with 0.1% BSA, 20mM HEPES (pH 7.4), and GloSensor reagent (Promega). cAMP-luminescence assays were performed following addition of a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX; 200μM) using a GloMax-Multi Detection System plate reader (Promega) as described previously [24 (link)].
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