Lysis and detection buffer

The medium was aspirated from each well, and the cells were washed with 1 ml of PBS. Cells were lysed by the addition of 1.5 ml of lysis and detection buffer (Cisbio) for 30 min. Subsequently, 16 µl aliquots of the lysate were added to the wells of a 384-well plate (PerkinElmer) in triplicates, together with 4 µl of pre-mixed HTRF antibodies (cAMP Gs dynamic kit, Cisbio) and the plate was sealed and incubated at room temperature for 1 h. The HTRF was measured at 620 nm and 665 nm using PheraSTAR® FSX plate reader (BMG Labtech) using the TR-FRET optical module. The HTRF ratio and cAMP concentration were calculated according to the cAMP Gs dynamic kit protocol.

Agonist-dependent inositol monophosphate (IP1) accumulation was measured using the IP-One G_q kit (Cisbio), a cell-based homogeneous time-resolved fluorescence assay, according to the manufacturer’s instructions. Briefly, cryo-preserved CHO cells stably expressing human OX₁R and human OX₂R were thawed, and resuspended in IMDM medium (Gibco) at densities of 2.0 × 10⁵ cells/ml and 4.0 ×10⁵ cells/ml, respectively. Cells were seeded into 384-well plates (50 µl per well) and incubated at 37 °C in 5% CO₂ for 20–24 h. In a separate plate, a dilution series of each agonist was prepared in Stimulation Buffer (Cisbio). The culture medium was removed and cells were incubated with 14 µl of agonist-containing Stimulation Buffer at 37 °C. After 1–2 h, 6 µl of Lysis and Detection Buffer (Cisbio) containing IP1-d2 and IP1 Tb cryptate antibody conjugates were added, and the reaction was incubated at room temperature protected from light. After 1 h, fluorescence was measured on an EnVision fluorescence plate reader (PerkinElmer) with the excitation wavelength set to 320 nm, and emission monitored at 620 nm (donor) and 665 nm (acceptor). Data were analyzed using GraphPad Prism.