Celllux

Cells were seeded at 50,000 cells/well in a poly-d-lysine coated 96-well black well/clear bottom plate (BD Falcon, Franklin Lakes, NJ, USA) and cultured overnight. After 16–24 h, cells were loaded with Fluo4-AM (Invitrogen, Waltham, MA, USA) for 30 min. Cells were then washed two times with assay buffer C1. After the final wash, a 100 µL residual volume remained on the cells. Peptides and other ligands were dissolved in 10% DMSO to a concentration of 1 mM and were diluted in C1 solution with 0.1% BSA. They were aliquoted as 2× solutions in 96-well plates and were transferred simultaneously by the robotic system within the imager from the ligand plate to the cell plate. Fluorescence was recorded with the fluorescence imaging plate reader CellLux (Perkin Elmer, Waltham, MA, USA) simultaneously in all wells at an excitation wavelength of 488 nm and emission wavelength of 510 nm in 1.5 s intervals over a period of 4 min. Fluorescence data were generated in duplicate, and experiments were repeated at least two times.

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.

Cells expressing different receptors were transfected with an expression vector encoding a receptor polypeptide and were allowed to grow until that receptor was expressed. A fluorescent probe (Fluo8 Direct; Invitrogen, Carlsbad, CA, USA) mixed with probenecid in HBSS/2 M HEPES buffer (pH 7.4) was added to each well and allowed to equilibrate with the cells for 1 h at 37 °C. Plates were then placed in a CellLux™ (PerkinElmer, Waltham, MA, USA) and the tested phlorotannins, reference agonist, or buffer (blank) were added and the fluorescence intensity was measured. Agonist or antagonist effects were calculated as the percentage or percent inhibition of the control response to a known reference agonist for each target, respectively.