cAMP Biosensor Assay in mLTC1 Cells

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).