cAMP is one of the most important second messengers, mediating diverse physiological responses of neurotransmitters, hormones, and drugs. The intracellular level of cAMP is determined by the balance between the activities of two enzymes: adenylyl cyclase (AC) which synthesizes cAMP from ATP, and phosphodiesterase (PDE) which degrades cAMP to AMP. The activity of AC is controlled through various G protein-coupled receptors (GPCRs) which are divided into two general classes depending on whether they stimulate or inhibit AC activity. The LANCE cAMP assay is a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay designed to measure cAMP levels produced upon modulation of adenylyl cyclase activity by GPCRs. We describe here a protocol for automating cell-based LANCE cAMP assays using cells expressing both classes of GPCRs using the JANUS Cellular Workstation.
The provides a complete, integrated automated system solution ideal for cellular assays in the areas of target identification and validation, assay development, secondary screening, and early ADME/Tox profiling and screening applications.
PerkinElmer's JANUS Cellular Workstation includes the JANUS Automated Workstation, the EnVision MultiLabel Reader, integrated labware movement and dynamic scheduling with incubation and platewashing options. The system also provides tested, pre-defined assay templates, documented results and application guides. Assay throughput may be quickly and easily scaled up 6 fold or greater as compared to manual methods. Results presented here demonstrate the key performance and pharmacological parameters of the assay with typical Z’ values between 0.80 and 0.90.
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