Spark fluorescence plate reader

Signaling assays were performed with receptor constructs harboring the wild-type helix 8 and the full-length C-terminus as well as the entire ICL3. Agonist-induced IP1 accumulation was measured in transiently transfected HEK293T/17 cells, as described before^{82 (link)}. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline (PBS), detached with trypsin-EDTA (Sigma), and resuspended in assay buffer (10 mM HEPES pH 7.4, 1 mM CaCl₂, 0.5 mM MgCl₂, 4.2 mM KCl, 146 mM NaCl, 50 mM LiCl, 5.5 mM glucose, 0.1% (w/v) BSA). Cells were seeded at 20,000 cells per well in white 384-well plates (Greiner) and incubated for 2 h at 37 °C with a concentration range of phenylephrine (Tocris) diluted in assay buffer. IP1 accumulation was measured using the HTRF IP-One kit (Cisbio) according to the manufacturer’s protocol on a SPARK fluorescence plate reader (Tecan). This kit uses an anti-IP1 antibody labeled with Tb-cryptate as FRET-donor (excitation at 317 nm, emission at 620 nm) and IP1-d2 as FRET-acceptor (emission at 655 nm). The ratio of emission intensities of FRET-acceptor and FRET-donor (Em655 nm/Em620 nm) was calculated, and EC₅₀ (median effective concentration) values were obtained by fitting the data with a three-parameter non-linear regression with GraphPad Prism Suite 8.4.3.

Signaling experiments were performed on whole cells with transiently transfected HEK293T cells as described before^{11 (link),14 (link)}. Twenty-four hours after transfection, cells were washed with PBS, detached with cell dissociation buffer (Gibco) and washed again in PBS. Cells were resuspended in assay buffer [10 mM Hepes (pH 7.4), 146 mM NaCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 4.2 mM KCl, 5.5 mM glucose, 50 mM LiCl, 1 mM 3-isobutyl-1-methylxanthin). cAMP and IP1 accumulation assays were performed on white low-volume 384-well plates (Greiner) using the cAMP Tb kit and the IP-One Tb kit (both from CisBio), respectively, according to the manufacturer’s protocol. For cAMP accumulation, 5000 cells were incubated with agonist at the indicated concentrations for 30 min at RT. To determine basal receptor signaling, cells were incubated in isobutylmethylxanthine (IBMX)-containing assay buffer for 30 min in the absence of ligand. For IP1 accumulation, 20,000 cells were incubated with agonist at the indicated concentrations for 2 h at 37 °C. Fluorescence intensities were measured on a Spark fluorescence plate reader (Tecan). To generate concentration-response curves, data were fitted to a three-parameter logistic equation.