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cAMP TR-FRET assay

NB Nisha Bhudia
SD Sapna Desai
NK Natalie King
NA Nicolas Ancellin
DG Didier Grillot
AB Ashley A. Barnes
SD Simon J. Dowell
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HEK 293 T cells were seeded into T25 flasks at 60,000 cells/cm2 and incubated at 37 °C with 5% CO2 overnight. Cells were transiently transfected for 48 hours in total, for the last 16 hours of incubation 100 ng/ml pertussis toxin was present in the media. Constitutive levels of cAMP accumulation were measured using the homogeneous, Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) LANCE cAMP kit (Perkin-Elmer, Waltham, MA, USA) as per manufacturer’s instructions. The assay was performed using 10,000 cells/well in white, low volume 384-well plates (Greiner Bio-One, Monroe, NC, USA) in the presence of 100 μM Rolipram and between 200–800 nM Forskolin pre-stimulation. The TR-FRET signal was read on a Viewlux imager (Perkin-Elmer) after a 3 hour room temperature incubation.

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