The cAMP-Glo Max Assay is a homogeneous, bioluminescent and high-throughput assay to measure cyclic AMP (cAMP) levels in cells. Compounds that modulate GPCRs coupled with adenylate cyclase typically alter intracellular cAMP levels. The cAMP-Glo Max Assay monitors cAMP levels in cells in response to the effect of agonists, antagonists or test compounds on G protein-coupled receptors (GPCRs). The assay is based on the principle that cyclic AMP (cAMP) stimulates protein kinase A (PKA) holoenzyme activity, decreasing available ATP and leading to decreased light production in a coupled luciferase reaction. This improved version combines the lysis and cAMP reaction buffers into the cAMP-Glo ONE Buffer. This new format streamlines the protocol and reduces the time needed to complete the assay. The new ONE Buffer is supplied at a 5X concentration, which provides increased flexibility for starting cell culture volumes. The cAMP-Glo Max Assay can be performed in 96-, 384- or 1536-well plates. The cells are induced with a test compound for an appropriate period of time to modulate cAMP levels. After induction, cells are lysed, and the cAMP released stimulates protein kinase A in the reagent (Figure 1). The Kinase-Glo Reagent is then added to terminate the PKA reaction and detect the remaining ATP via a luciferase reaction. Plates are read using a microplate-reading luminometer. The half-life for the luminescent signal is greater than 4 hours providing ample time to read the plates and eliminating the need for luminometers with reagent injectors.
