Development of a novel cell-based, In-Cell Western/ERK assay system for the high-throughput screening of agonists acting on the delta-opioid receptor (original) (raw)

Background: Extracellular signal-regulated kinases (ERKs) are important signaling mediators in mammalian cells and, as a result, one of the major areas of research focus. The detection and quantification of ERK phosphorylation as an index of activation is normally conducted using immunoblotting, which does not allow high-throughput drug screening. Plate-based immunocytochemical assays provide a cheaper and relatively high-throughput alternative method for quantifying ERK phosphorylation. Here, we present optimization steps aimed to increase assay sensitivity and reduce variance and cost using the LI-COR In-Cell Western (I-CW) system in a recombinant CHO-K1 cell line, over-expressing the human delta-opioid receptor (hDOPr) as a model. Methods: Cells cultured in 96-well microassay plates were stimulated with three standard/selective DOPr agonists (SNC80, ADL5859, and DADLE) and a novel selective DOPr agonist (PN6047) to elicit a phospho-ERK response as an index of activation. A number of experimental conditions were investigated during the assay development. Key results: Preliminary experiments revealed a clearly visible edge-effect which significantly increased assay variance across the plate and which was reduced by pre-incubation for 30 min at room temperature. ERK phosphorylation was detectable as early as 1 min after agonist addition, with