Pdl coated white 96 well cell culture plate with solid flat bottom

6 × 10⁵ cells ml⁻¹ ΔFZD_1-10 HEK293 T cells were seeded onto PDL-coated white 96-well cell culture plate with solid flat bottom (Greiner Bio-One). Next day, cells were transfected with 20 ng of SNAP-tagged receptor, 20 ng M50 Super 8× TOPFlash (Addgene, 12456), 2 ng pRL-TK Luc (Promega, E2241) and 58 ng pcDNA plasmid DNA to a final amount of 100 ng of plasmid DNA per well. Four hours after transfection, medium was changed to starvation medium (DMEM without FBS) containing either vehicle or 1000 ng/ml recombinant WNT-3A for FZD transfected cells or 100 nM SAG1.3 for SMO-transfected cells. Twenty-four hours after stimulation, cells were lysed gently shaking with 20 µl 1× Passive Lysis Buffer (Promega, E1910) for 15 min. Subsequently, 20 µl of LAR II (Promega, E1910) were added to all wells after which luminescence (580-80 nm) was read and then 20 µl of Stop & Glo (Promega, E1910) were added to all wells after which luminescence (480-80 nm) was read again with a CLARIOstar microplate reader (BMG). Data were analyzed using GraphPad Prism 6.

ΔSMO HEK293 cells were transiently transfected in suspension using Lipofectamine 2000 (Thermo Fisher Scientific). For the experiments 4 × 10⁵ cells ml⁻¹ were transfected with 100 ng of Gα_i1–LgBiT plasmid DNA, 500 ng of SmBiT–Gβ₅, 500 ng of Gγ₂, and 200 ng of receptor plasmid DNA. The cells (100 µl) were seeded onto a PDL-coated white 96-well cell culture plate with solid flat bottom (Greiner Bio-One). Forty eight hours post transfection, the cells were washed once with 0.1% BSA/HBSS (Gibco or Thermo Fisher Scientific) and maintained in the same buffer. The cells were stimulated with ligands 30 min after the addition of the luciferase substrate coelenterazine h (10 µM final concentration). Nluc_lum (470–80 nm) was measured using a CLARIOstar microplate reader (BMG). Data from the luminescence measurements obtained 5 min after the ligand addition were analyzed using GraphPad Prism 6.