Dmem buffer

Briefly, CHO-V_1AR cells were separately suspended in DMEM buffer (Invitrogen, Carlsbad, CA, USA) complemented with 0.1% FCSd and distributed into microplates (4.5× 10⁴ cells/well). Then, fluorescent probe (Fluo4, Invitrogen) mixed with probenecid in HBSS buffer (Invitrogen, Carlsbad, CA, USA) supplemented with 20 mM HEPES, pH 7.4 (Invitrogen) was added to each well, allowing to equilibrate with the cells for 60 min at 37 °C, then 15 min at 22 °C. Thereafter, the assay plates were positioned in a CellLux microplate reader (PerkinElmer, Waltham, MA, USA) and dieckol and PFF-A (12.5, 25, 50, 100, and/or 150 μM), reference agonist, or HBSS buffer (basal control) was added. For stimulated control measurements, AVP at 1 µM was added in separate assay wells. The agonist effect on V_1AR was calculated as a % of control response to 1 μM AVP. Similarly, for the antagonist effect, % inhibition of the control response to 10 nM AVP was evaluated. AVP and [d(CH₂)₅¹, Tyr (Me)₂]-AVP were used as reference agonist and antagonist, respectively.

The functional assay was run onto cells expressing the hMCH-R1 receptor, as described in Wang et al. [61 (link)]. In brief, the cells are suspended in DMEM buffer (Invitrogen), then distributed in microplates at a density of 4.104 cells/well. The fluorescent probe (Fluo4 Direct, Invitrogen) mixed with probenecid in Hanks’ Balanced Salt solution (HBSS) (Invitrogen) complemented with 20 mM HEPES (Invitrogen) (pH 7.4) is then added into each well and equilibrated with the cells for 60 min at 37 °C, then 15 min at 22 °C. Then, the assay plates are positioned in a microplate reader (CellLux, PerkinElmer) which is used for the addition of the test compound, reference agonist or HBSS buffer (basal control), and the measurements of changes in fluorescence intensity, which varies proportionally to the free cytosolic Ca²⁺ ion concentration. For stimulated control measurements, MCH at 300 nM is added in separate assay wells. The results are expressed as a percentage of the control response to 300 nM MCH. The standard reference agonist is MCH, which is tested in each experiment at several concentrations to generate a concentration-response curve from which its EC₅₀ value is calculated. For the antagonist effect, the results are expressed as a percentage inhibition of the control response to 30 nM MCH, which was added to all tested wells and controls 5 min after the candidate products.

In general, transfected HEK-293 cells suspended in DMEM buffer (Invitrogen) containing 1% FCSd were distributed in microplates at a density of 2.10⁴ cells/well. Then, the mixture of fluorescent probe (Fluo4 Direct, Invitrogen) and probenecid in HBSS buffer (Invitrogen) complemented with 20 mM Hepes (Invitrogen) (pH 7.4) was added into each well and incubated for 60 min at 37 °C, followed by 15 min incubation at 22 °C. Thereafter, the assay plates were positioned in a CellLux microplate reader (PerkinElmer, Waltham, MA, USA) which was used for the addition of the following: For agonist assay—test compound, reference agonist, or HBSS buffer (basal control). Linoleic acid at 100 µM was added in separate assay wells for stimulated control measurement. For antagonist assay—test compound or HBSS buffer (basal and stimulated control), then, 5 min later, 20 µM linoleic acid. Agonist results are expressed as a percent of the control response to 100 µM linoleic acid while antagonist results are expressed as percent inhibition of the control response to 20 µM linoleic acid.