CHEMBL5730041

pERK High-Content Assay: Cell Culture: Chinese Hamster Ovary (CHO) cells stably expressing the hD2L dopamine receptor (Urban et al., Neuropsychopharmacology 32:67 (2007)) were maintained in Ham's F12 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 ug/mL streptomycin, and 0.5 ug/ml G418. On day 1 of the assay, cells were seeded onto black clear-bottom tissue culture-treated 96-well plates (Greiner, BioOne). On day 2 of the assay, cells were washed with serum-free medium (Ham's F-12, penicillin and streptomycin) and incubated in 100 uL serum-free medium overnight.Immunofluorescence: Automatic multichannel pipetters were used for liquid handling and multichannel vacuum manifolds for aspirations. Each tested concentration was typically measured in triplicate or quadruplicate. For concentration curves, half-log-dilutions were used. Drug dilutions were prepared in stimulation medium (serum-free medium, 100 mg/L ascorbic acid).