Freestyle 293 suspension media

BRET assays were performed and analyzed as previously described 4 (link) with the following modifications: HEK-293S cells grown in FreeStyle 293 suspension media (Thermo Fisher) were transfected at a density of 1 million cells/mL in 2 mL volume using 600 ng total DNA at 1:1 ratio of Receptor-rLuc:Nb6-mVenus and a DNA:PEI ratio of 1:5, and incubated in a 24 deep well plate at 220 rpm, 37°C for 48 hours. Cells were harvested by centrifugation, washed with Hank's Balanced Salt Solution (HBSS) without Calcium/Magnesium (Gibco), and resuspended in assay buffer (HBSS with 20 mM HEPES pH 7.45) with 1 μg/mL freshly prepared coelenterazine h (Promega). Cells were plated in white-walled, white-bottom 96 well plates (Costar) in a volume of 60 μl/well and 60,000 cells/well. Ligands were prepared in drug buffer (assay buffer with 0.1% BSA, 6 mM CaCl2, 6 mM MgCl2), and added at a 1:2 ratio of drug:cell suspension. Ten minutes after the addition of ligand, plates were read using a SpectraMax iD5 plate reader using 485 nm and 535 nm emission filters with a one-second integration time per well. The computed BRET ratios (mVenus/RLuc emission) were normalized to ligand-free control (Net BRET) prior to further analysis.