“…The compound library screenings and the subsequent functional characterization of the analogues in fluorescence-based Ca 2+ imaging assays using the Fluo-4/AM (α3β4, α6/α3β4β3, and α7) or Fluo-8/AM (α4β2 and α6/α3β2β3 V9’S ) dyes were performed essentially as previously described. , Briefly, the nAChR-HEK293 cells were split into poly- d -lysine-coated black 96-well plates with a clear bottom (0.8–1.0 × 10 5 cells/well). The following day, the culture medium was aspirated and the cells were incubated in 50 μL of loading buffer [Hank’s Balanced Salt Solution (HBSS) containing 20 mM HEPES, 1 mM CaCl 2 , 1 mM MgCl 2 , and 2.5 mM probenecid, pH 7.4] supplemented with 4 mM Fluo-4/AM or Fluo-8/AM at 37 °C for 1 h. Then, the loading buffer was aspirated and the cells were washed once with 100 μL of assay buffer [140 mM N -methyl- d -glucamine (NMDG), 5 mM KCl, 1 mM MgCl 2 , 10 mM CaCl 2 , 20 mM HEPES, and 2.5 mM probenecid, pH 7.4, for α4β2 and α6/α3β2β3 V9’S , α6/α3β4β3, and α3β4 nAChRs; HBSS containing 20 mM HEPES, 1 mM CaCl 2 , 1 mM MgCl 2 , and 2.5 mM probenecid, pH 7.4, for α7] (the assay buffer for α7 was supplemented with 5 μM PNU-120596).…”