Phospholipase C (PLC) activation was measured via the luciferase activity of a reporter gene (NFAT-luc, Promega, Madison, USA) as previously described (Cheng et al., 2010 (link)). HEK293 were co-transfected with a plasmid containing the NFAT and firefly luc reporter gene (NFAT-luc, pGL4.33), together with either receptor or empty vector plasmid DNA (mock transfection) in an equimolar concentration (0.45 μg plasmid per well). The thyrotropin (TSH) receptor served as a positive control and was stimulated with 100 mU/ml bovine TSH (Sigma-Alderich, St. Louis, MO, USA) (Winkler et al., 2010 (link)). For the PTX assay ADRA2A as Gi coupled receptor was used as positive control.
Forty-eight hours post transfection, cells were incubated for 6 h with 3-T1AM and/or 5-HT in supplement-free MEM at 37°C with 5% CO2. The media was removed and cells lysed for 15 min on a shaking platform at room temperature using 50 μl of 1x passive lysis buffer (PLB, Promega, Madison, USA). To determine luciferase activity, 10 μL sample were transferred to a black 96-well plate. Forty microliter luciferase substrate (Promega, Madison, USA) were automatically injected using the Berthold Microplate Reader and immediately measured.
Forty-eight hours post transfection, cells were incubated for 6 h with 3-T1AM and/or 5-HT in supplement-free MEM at 37°C with 5% CO2. The media was removed and cells lysed for 15 min on a shaking platform at room temperature using 50 μl of 1x passive lysis buffer (PLB, Promega, Madison, USA). To determine luciferase activity, 10 μL sample were transferred to a black 96-well plate. Forty microliter luciferase substrate (Promega, Madison, USA) were automatically injected using the Berthold Microplate Reader and immediately measured.