Snap lumi4 tb

Cells stably or transiently expressing SNAP-GLP-1R were dual-labeled with the TR-FRET probe SNAP-Lumi4-Tb (40 nM, Cisbio) and SNAP-Surface 647 (1 μM, New England Biolabs) for 60 min at room temperature. These concentrations were selected because they provided optimum signal intensity at both measurement wavelengths. Where relevant, MβCD treatments were performed after labeling. Cells were washed and placed in HBSS in a white plate for a 10-min baseline measurement at 37 °C using a Spectramax i3x (Molecular Devices) plate reader fitted with a TR-FRET filter set (λEx = 335 nm, λEm = 616 nm and 665 nm, delay 30 μs, integration time 400 μs). TR-FRET was sequentially measured after agonist addition. The ratio of fluorescent signals at both emission wavelengths (665/616) was considered indicative of clustering because it reflects both transient and stable interactions between receptor protomers occurring within the long fluorescence lifetime of the excited terbium cryptate. Dose-response curves were constructed from AUC obtained from kinetic traces at different agonist concentrations and fitted to 4-parameter logistic curves to calculate potency (log EC₅₀) and efficacy (E_max) values.

Ligand-binding experiments were performed on whole cells for comparison of affinities for wild-type and receptor mutants using an HTRF binding assay as previously described^{17 (link)}. Forty-eight hours post-transfection the cells were labelled with 50 nM SNAP-Lumi4-Tb (Cisbio) in labelling buffer (20 mM Hepes (pH 7.5), 100 mM NaCl, 3 mM MgCl₂, and 0.05% (w/v) bovine serum albumin (BSA)) for 1.5 h at 37 °C. The cells were washed two times with labelling buffer and two times with assay buffer (20 mM Hepes (pH 7.5), 100 mM KCl, 3 mM MgCl_2, and 0.05% (w/v) BSA)) and subsequently incubated for 1 h at room temperature with a concentration range of fluorescently labelled peptide HiLyte Fluor 488-Orn⁸ oxytocin (Eurogentec) in assay buffer. Fluorescence intensities were measured on a Spark fluorescence plate reader with an excitation wavelength of 340 nm and emission wavelengths of 620 nm and 510 nm for Tb³⁺ and the fluorophore HiLyte Fluor 488, respectively. The ratio of fluorescence resonance energy transfer (FRET) donor and acceptor fluorescence intensities was calculated (F510 nm/F620 nm). Nonspecific binding was determined in the presence of a 1000-fold excess of unlabelled oxytocin. Data were analysed by global fitting to a one-site saturation binding equation with the GraphPad Prism software.

HEK293 cells (ATCC, CRL-1573, lot: 3449904) were cultivated in DMEM (Thermo Fischer Scientific, Courtaboeuf, France) complemented with 10% (v/v) fetal bovine serum. Absence of mycoplasma was routinely checked using the MycoAlert Mycoplasma detection kit (LT07-318, Lonza, Amboise, France), according to the manufacturer protocol. All drugs (DCG-IV, LY341495, LY379268, and LY487379) were from Tocris Bioscience (Bristol, UK). LSP4-2022 was provided by Dr. F. Acher (Paris, France). All HTRF reagents, labeled monoclonal antibodies anti-c-Myc-d2 (61MYCDAA, Cisbio Bioassays, Codolet, France), and anti-6His-d2 (61HISDLA, Cisbio Bioassays), labeled ligands (SNAP-Lumi4-Tb, SNAP-Red, CLIP-Red, and BG-Alexa Fluor 488) were a kind gift from Cisbio Bioassays. The pRK5 plasmids encoding wild-type rat mGluR subunits, with a HA-tag and with SNAP or CLIP inserted just after the signal peptide, were previously described^{46 (link)}. pEGFP-C2 plasmid encoding EGFP was from Clontech (Mountain View, CA, USA). Point mutations were introduced in the SNAP-tag mGlu2 or mGlu3 plasmids according to the QuikChange mutagenesis protocol (Agilent Technologies, Santa Clara, CA, USA).

Ligand binding was performed with a homogeneous time-resolved fluorescence-based assay. N-terminal-SNAP-tagged ghrelin receptor (WT or with various mutations) and full-length ghrelin labeled with the dye A2 on an additional cysteine at the C-terminal end of the peptide (ghrelin-A2, synthesized by Vazyme, China) were used as previously described^{41 (link)}.
HEK293 cells transfected with SNAP-ghrelin receptor (WT or mutants) were seeded at a density of 1 × 10⁶ cells into 3 cm dish and incubated for 24 h at 37 °C in 5% CO₂. Cell culture medium was removed and Tag-lite labeling medium with 100 nM of SNAP-Lumi4-Tb (Cisbio, SSNPTBC) was added, and the cells were further incubated for 1 h at 37 °C in 5% CO₂. The excess of SNAP-Lumi4-Tb was then removed by washing 4 times with 1 ml of Tag-lite labeling medium.
For saturation binding experiments, we incubated cells with increasing concentrations of ghrelin-A2 in the presence or absence of 10 μM unlabeled ghrelin for 1 h at R.T. Signal was detected using the Multimode Plate Reader (PerkinElmer EnVision) equipped with an HTRF optic module allowing a donor excitation at 340 nm and a signal collection both at 665 nm and at 620 nm. HTRF ratios were obtained by dividing the acceptor signal (665 nm) by the donor signal (620 nm). Kd values were obtained from binding curves using Prism 8.0 software (GraphPad Software).

Cells were labelled with SNAP-Lumi4-Tb (Cisbio) using 40 nM probe for 60 min at 37 °C, in complete medium. After washing to remove unbound probe, cells were resuspended in HBSS with 0.1% BSA and metabolic inhibitor cocktail (20 mM 2-deoxyglucose and 10 mM NaN₃ to inhibit endocytosis, as previously described [9] (link), [23] (link), and seeded into white opaque plates. After 20 min at room temperature, cells were then placed at 4 °C, and a range of concentrations of test ligands were applied concurrently with 10 nM Luxendin645 [19] (link) or 5 nM exendin-4-Cy5, with a range of concentrations of Luxendin645 or exendin-4-Cy5 also applied to establish equilibrium binding parameters for the competing labelled GLP-1R probe. After a 24-hour incubation period at 4 °C, binding was measured by TR-FRET using a Spectramax i3x plate reader (Molecular Devices) fitted with an HTRF module.

HEK293‐SNAP‐GLP‐1R cells were labelled in suspension with SNAP‐Lumi4‐Tb (40 nM, Cisbio, Codelet, France) for 1 hour at room temperature in complete medium. After washing and resuspension in hanks' balanced salt solution containing 0.1% bovine serum albumin and metabolic inhibitors (20 mmol/L 2‐deoxygucose and 10 mmol/L NaN₃) to prevent GLP‐1R internalization,¹³ binding experiments were performed by time‐resolved förster resonance energy transfer (FRET) using exendin (9‐39) with fluorescein isothiocyanate (FITC) installed at position K12 as previously described.¹⁴ For further details, see Supplementary Methods.

CHO cells were transfected according to the manufacturer's recommendation (jetPEI DNA transfection, Polyplus-transfection, Illkirch, France). Briefly, cells were seeded on day 1 in 6-well plates at a concentration of 300,000 cells/well. On day 2, 6 μl/well of JetPEI diluted in 100 μl NaCl (150 mM) were added to a mix of DNA coding for SNAP-OXTR (180 ng/well) and uncoding DNA (2820 ng /well) diluted in 100 μl NaCl (150 mM). The mixture was incubated for at least 30 min at room temperature and was then added onto the cells. On day 3, cells in the 6-well plates were then harvested after addition of trypsin, counted and seeded at a concentration of 30,000 cells/well in a white 96-well plates. Experiments were carried out on day 4. Cells were labeled with SNAP-Lumi4-Tb (100 nM) (Cisbio Bioassays, Codolet, France) at 37°C for 1 hour, rinsed four times with Tag-lite buffer (Cisbio Bioassays) and incubated in the presence of the various compounds. The time-resolved FRET (TR-FRET) signal was measured on a Pherastar (BMG Labtech). Cells were illuminated at 337 nm, and luminescent signals were measured at 620 nm and 665 nm every minute. The ratio (665/620) was then plotted as a function of time. Experiments were performed three times, independently.