Fig. 5.

Assay optimization. (A) HEK293-CNG cells stably expressing the Gal3/1ctR chimera were plated at 5000 (●), 10,000 (■), 15,000 (▲), or 20,000 (▼) cells per well in a 384-well plate. After 18-h incubation at 37°C, 5% CO₂, cells were loaded with ACT:One membrane potential dye for 2 h at room temperature. cAMP levels were determined in response to increasing concentrations of porcine galanin in the presence of an EC₉₀ of forskolin (1 μM) according to ACT:One manufacturer's instructions and as described in detail in Table 1. The optimal cell density was determined to be 10,000 cells per well. At this density signal-to-background ratio (S:B) was 2.9. (B) HEK293-CNG cells stably expressing the Gal3/1ctR chimera were plated at 10,000 cells per well and cAMP levels at increasing concentrations of porcine galanin were determined as in (A) in the presence of an EC₉₀ of forskolin (1 μM) and in the presence of 0.025% (●), 0.5% (■), 0.8% (▲), 1.5% (▼), or 2% (♦) DMSO. The Gal3/1ctR cAMP assay performance, both in terms of S:B and galanin EC₅₀ values, was not significantly affected by DMSO concentrations up to 2%. Final DMSO concentration used in the high-throughput screening (HTS) assay was 0.7%. The data presented are means±SEM of triplicate wells (n=3).