Mithras lb 943 plate reader

5.10⁴ HEK293FT cells were co-transfected with Rluc-tagged receptors and YPet-β-arrestin2 (50–75 ng) eventually in presence of increasing amounts of GPR88 (0–75 ng) in white 96-well plates (Greiner Bio-One). Each transfection was duplicated in black 96-well plates (Greiner Bio-One) in order to measure the expression level of YPet-β-arrestin2. 36 hr after transfection, cells were starved for 16 hr. In black plates, cells were incubated in PBS containing 5 mM HEPES and YPet fluorescence was measured at 540 nm (excitation 480 nm) using the Mithras LB 943 plate reader (Berthold). Net YPet fluorescence was calculated as the sample fluorescence minus the fluorescence of the Rluc-only sample of each experiment. For BRET, white plates were stimulated with vehicle or agonists in PBS containing 5 mM HEPES and 5 μM coelenterazine H and emission at 480 nm (Rluc8 bioluminescence) and at 540 nm was immediately measured for 35 min using the Mithras LB 943 plate reader (Berthold). BRET ratios (540 nm emission/480 nm emission) were calculated and net BRET ([BRET] - [BRET in absence of Venus acceptor]) was normalized to the YPet fluorescence signal intensity. Ligand-induced BRET changes were calculated as the difference between basal and agonist values, expressed as a percentage of the maximal response (absence of GPR88) and plotted as a function of the amount of GPR88 cDNA transfected.

For the end-point dose-response experiments, medium was aspirated and cells were re-suspended in 40 µl/well of PBS 1X, 1 mM HEPES. Cells were incubated for 30 minutes at 37 °C in a total volume of 40 µl/well of PBS 1X, 1 mM HEPES containing or not increasing concentrations of rhCG and rhLH. BRET measurements were performed upon addition of 10 µl/well of 5 µM Coelenterazine h (Interchim, Montluçon, France), using Mithras LB 943 plate reader (Berthold Technologies GmbH & Co. Wildbad, Germany). For the real-time kinetics, cells were re-suspended in 60 µl/well of PBS 1X, 1 mM HEPES and, for cAMP kinetics, 200 µM IBMX. BRET measurement was immediately performed upon addition of 20 µl/well of EC₅₀ concentrations of rhCG and rhLH as previously calculated in dose-response experiments, and 20 µl/well of Coelenterazine h.

Testosterone levels were measured using an HTRF-based assay Kit (CisBio Bioassays, Codolet, France) using the same method as in our previous work [30 (link)]. Primary Leydig cells in serum-free RPMI were cultured in 96-well plates at 120,000 cells/well and then incubated for 1 h with or without KH7, 2-CE, 4-CE, LRE1, or ddAdo5′. They were then stimulated with hLH (0.7 nM), or only with serum-free RPMI (control) for 3 h at 35 °C. Afterwards, 10 µL of culture supernatant were transferred to a 384-well white microplate and 5 µL of testosterone-XL665 + 5 µL of anti-testosterone-Ey3 + cryptate antibody were added. The 384-well microplate was then incubated at room temperature in the dark for 1 h, excited with a Mithras LB 943 plate reader (Berthold Technologies GmbH & Co. Wildbad, Germany) at 320 nm, and finally fluorescence was measured at 620 nm and 665 nm.