The use of frozen cells for cell based screening has become widely accepted within the drug discovery community. Separating cell production from the actual screening campaign not only  increases your flexibility but also improves the data consistency as the cellular material can be controlled and validated before running the functional assay. One of the methods used to deliver frozen cells as a consumable to the final user is to gamma irradiate the cells, so that cells cannot resume growth after thawing.

This material is currently available for GPCR-expressing cells: AequoZen®cells, validated for calcium flux assays (AequoScreen®or fluorescence assays) and cAMPZen®cells, validated for the LANCE®cAMPassay.

The increasing amount of evidence for biased agonism(collateral efficacy and varying potencies according to the signal transduction pathway observed), and the search for more physiologically relevant recording of the activity of drugs in development, results in an increasing demand for assays relating to other signaling pathways activated by GPCRs. Amongst these GPCR-triggered pathways are numerous kinase cascades, including MAP Kinase which leads to ERK activation. The location of this kinase downstream from the activation of many GPCRs makes measuring the phosphorylationideal for evaluating pathway activation/inhibition in the presence of small molecules. AlphaScreen®SureFire®is a homogenous assay format for measuring ERK phosphorylationin cells. In this assay system, activated ERK binds a combination of two antibodies, one of which can only bind when ERK is phosphorylated. Only modified proteins that bind both antibodies are detected, using the Alpha technology (PerkinElmer) containing streptavidin coated Donor beads and Protein A coated Acceptor beads.

We show here that commercially available frozen, gamma-irradiated cAMPZen cells can be used together with ERK, MEK and Akt AlphaScreen®SureFire®assays to assay GPCR stimulation of the MAP Kinase pathway.