Furimazine stock solution

For BRET measurements with the Nluc-FZD_5/6 CRD sensors, 15,000 HEK293T cells were plated in PDL-precoated white 96-well plates. Next day, cells were transfected with 0.09 μg of the indicated constructs and 0.01 μg of tRNA/synthetase and were cultured in the presence of 0.1 mM TCO*K. Forty-eight hours after transfection, cells were washed in DPBS, kept for 2 hours in DMEM, and labeled with 1 μM Tet-Cy3 or Tet–BDP-FL for 30 min. Cells were washed with DPBS and kept for an additional 30 min in DMEM. Next, cells were again washed with DPBS and incubated with 90 μl of a 1/1000 dilution of furimazine stock solution (Promega) in 0.1% BSA/HBSS. After 5 min of incubation, the basal BRET ratio was measured in three consecutive reads, and 10 μl of a WNT-3A or WNT-5A solution (3 μg/ml; in 0.1% BSA/HBSS) or vehicle control was applied per well. Subsequently, the BRET ratio was recorded for an additional 25 to 60 min. For experiments with a higher temporal resolution, eight baseline BRET reads were recorded within 2 min prior manual addition of compounds or vehicle control, followed by at least 40 reads. All experiments were conducted using a CLARIOstar plate reader (BMG Labtech, Ortenberg, Germany) equipped with monochromators to separate Nluc (450/80 nm), BDP-FL (520/40 nm), and Cy3 (580/30 nm), respectively.

G protein activation experiments with the G-CASE biosensors were conducted as previously described (Reference to PMID: 34516756). Briefly, transfected cells grown for 48 h in 96-well plates were washed with Hanks′ Balanced Salt solution (HBSS) and incubated with 1/1,000 dilution of furimazine stock solution (Promega, WI, USA). After incubation for 2 min, BRET was measured in three consecutive reads followed by addition of agonist (histamine/amthamine) solutions or vehicle control and subsequent BRET reads. All experiments were conducted at 37 °C. Nluc emission intensity was selected using a 470/80 nm monochromator and cpVenus emission using a 530/30 nm monochromator in a CLARIOstar plate reader with an integration time of 0.3 s.