*4.4. Functional Analysis of ORs Using the CellKeyTM System*

The analysis was performed as described previously [52]. In brief, cells were seeded at a density of 5.0 <sup>×</sup> <sup>10</sup><sup>4</sup> in CellKeyTM poly-D-Lysine (Sigma Aldrich, Saint Louis, MO, USA) coated 96-well microplates with an embedded electrode at the bottom of each well and incubated for 24 h. After washing with CellKeyTM buffer composed of Hanks' balanced salt solution (1.3 mM CaCl2·2H2O, 0.81 mM MgSO4, 5.4 mM KCl, 0.44 mM KH2PO4, 4.2 mM NaHCO3, 136.9 mM NaCl, 0.34 mM Na2HPO4, and 5.6 mM d-glucose) containing 20 mM 4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 0.1% bovine serum albumin (BSA), cells were incubated for 30 min at 28 ◦C, and then treated with vehicle or one of the reagents. The change in impedance of an induced extracellular current (dZiec) in each well was measured for 25 min, following a 5 min baseline measurement. The magnitude of change in the dZiec value was defined as ∆Ziec, and the value for rubiscolins was calculated as a percentage using the highest value for each positive control.
