Lance camp 384 kit

HRMECs were cultured and grown at a density of 1 × 10⁴ cells per well in 96-well plates. After overnight serum starvation, the cells were reacted with 10 μM forskolin (Sigma-Aldrich, St. Louis, MO, USA) and 0.5 mM IBMX (Sigma-Aldrich) in the presence of compounds for 10 min at 37 °C and then treated with lysis buffer (50 mM HEPES, 10 mM CaCl₂, 0.35% Triton X-100). The cAMP concentration in cell lysates was determined by LANCE cAMP 384 kit (PerkinElmer, Shelton, CT, USA) or LANCE Ultra cAMP Kit (PerkinElmer) following the manufacturer’s instructions. The EC₅₀ value was calculated via nonlinear regression by GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA).

A LANCE cAMP384 kit (PerkinElmer, MA, USA) was used for cAMP quantification. Cells were seeded onto 24-well plates. The next day, growth medium was replaced with serum-free with 10 µM morphine (Shenyang First Pharmaceutical Factory, China) for 6, 12, 16 or 24 h, then harvested with Versene dissociation solution (Invitrogen) and washed with Hanks’ balanced salt solution (HBSS) 1 × buffer (Invitrogen). The cells were then resuspended at a concentration of 2 × 10⁶ cells/mL in stimulation buffer [HBSS containing 5 mM HEPES (Sigma, St. Louis, MO, USA), 0.1% bovine serum albumin, 0.05 mM IBMX (Sigma)]. Alexa Fluor 647-labeled antibodies were added to the final cell suspension, and then 100 µM naloxone (Sigma) was added to precipitate the cAMP overshoot. After incubation at 37 °C for 15 min, Detection Mix(including in the kit) was added to the mixture. The sample was further incubated for 1 h, and LANCE signal was measured on an Infinite F200 microplate reader (Tecan, Grödig, Austria). A cAMP standard curve was constructed and cellular cAMP level was assayed simultaneously, according to the manufacturer’s instructions. The LANCE signal obtained at 665 nm was used to analyze cAMP levels directly. The signal at 615 nm was used to identify dispensing or quenching problems.

ThP-1 cells were incubated with 0.5 or 1 nM hSTC1-Flag in HBSS buffer containing IBMX for 45 min. Forskolin (FSK), a cAMP activator, was used as a positive stimulant. Cyclic AMP levels were measured using the Lance cAMP 384 kit (PerkinElmer Life Sciences). With the VICTOR X4 Multilabel Plate Reader (PerkinElmer Life Sciences), the fluorescent signal was measured at 340 nm and 615 and 665 nm (Fig S4C).

The day prior to the experiment cells were seeded in a 96-well tissue culture treated plate (Corning, NY, U.S.A.) at a density of 20,000 cells per well. The next day culture media was replaced with 50 µl of cAMP assay media (HBSS buffer with 0.1% w/v fatty acid-free BSA, 5 mM HEPES, and 0.5 mM IBMX, pH 7.4) and incubated at 37°C for 30 min to minimise basal cAMP levels. RXFP4 agonists and forskolin (positive control) were diluted in cAMP assay media. Once the 30-min incubation was complete, 25 µl of agonists or vehicle (0.01% DMSO) were added to each well and incubated for 15 min at 37°C. Afterwards 25 µl of forskolin dilutions was added to each well and incubated for further 15 min under same conditions. Following incubation, the contents of each well were aspirated and 50 µl of ice-cold ethanol was added and plates frozen for 15 min to extract cAMP. The plates were taken out from the freezer and kept in a fume hood to allow evaporation of ethanol after which 50 µl of cAMP detection buffer (0.35% triton X-100, 50 mM HEPES, 10 mM calcium chloride, pH 7.4) was added to each well and shaken gently at room temperature for 15 min. This cell lysate was used to quantify cAMP using the LANCE™ cAMP 384 kit (PerkinElmer, U.S.A. #TRF0262).

Functional assays were conducted to evaluate the activity of test compounds for the MC4R in transfected CHO cells by measuring cAMP release using a time-resolved fluorescence resonance energy transfer (TR-FRET) method (Eurofins Panlabs Item 332270). Commercially available frozen, irradiated CHO-K1 cells with transfected human recombinant melanocortin MC4 receptor were used (PerkinElmer Part ES-191-AF). Test compound and/or vehicle was incubated with the cells (2.5 x 10E5/ml) in modified HBSS pH 7.4 buffer at 37°C for 20 minutes. The reaction was evaluated for cAMP levels by TR-FRET using a commercially available kit (PerkinElmer LANCE™ cAMP 384 kit). Test compound-induced cAMP increase by 50 percent or more (≥50%) relative to the 3 μM NDP a MSH control response indicated possible receptor agonist activity. Test compound-induced inhibition of the 10 nM NDP-a-MSH induced cAMP response by 50 percent or more (≥50%) indicated possible receptor antagonist activity. Test compounds, including concurrent known agonist and antagonist controls, were run at 6 concentrations selected based on biochemical binding assay results to determine the IC₅₀.