Coelenterazine h

For cAMP analysis, 2 × 10⁴ mLTC1 were seeded in a 96-well plate and transiently transfected with 100 ng/well of the previously described cAMP biosensor Camyel-expressing vector^{53 (link),54 (link)} mixed with 0.5 μl/well of Metafectene PRO (Biontex Laboratories GmbH, München, Germany) in serum-free medium. After 48 h, the medium was removed and 40 μl/well of DPBS added of 1 mM HEPES (ThermoFisher Scientific) and 500 µM of IBMX were administrated to the cells for 20 min. Cells were then exposed to LH (1500 pM) and hCG (300 pM), 50 µM Forskolin or CTX in the range of 0.1–100 µg/ml, in the presence or in the absence of 1 nM and 1 µM BaP for 30 min. Please, note that forskolin could not serve as a proper positive control for cAMP production in the mLTC1 cell line, due to low efficacy in this model^{55 (link)}. Eventually, 5 µM coelenterazine H (Interchim, Montluçon, France) was added. Emissions from donor and acceptor were detected at wavelengths of 480 ± 20 and 540 ± 20 nm, respectively using the CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany).

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.

Transiently transfected cells were washed and preincubated at 37 °C and 5% CO₂ for 20 min in 40 µL of phosphate buffered saline (PBS) added with 1 mM HEPES and 500 µM of the phosphodiesterase inhibitor 3-isobutil-1-methylxanthine (IBMX). Then, cells were washed by PBS and maintained for 30 min at 37 °C and 5% CO₂ in 50 µL of PBS and 1 mM HEPES, in the presence or absence of increasing doses of LH or hCG (1 pM–100 nM range) [10 (link)]. Cells treated with 50 µM forskolin (Sigma-Aldrich) served as positive controls [31 (link)]. Bioluminescence resonance energy transfer (BRET) measurements were performed upon addition of 10 µL of 5 µM coelenterazine H (Interchim, Montluçon, France). Signals emitted by donor and acceptor tags were detected by the CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany) at wavelengths of 480 ± 20 and 540 ± 20 nm, respectively, and represented as a ratio (BRET changes) [32 (link)].