Measuring dynamic and real-time cAMP levels using cADDis

Paul Tewson, Scott Martinka, Catherine Berlot, Thom Hughes, Anne Marie Quinn & Shane Tillo

Real-Time cAMP Detection

Here, we introduce a new fluorescent cAMP sensor, cADDis, capable of detecting dynamic changes in cAMP concentration in living cells. We have applied cADDis to use in a novel Gi assay that, for the first time, directly reports Gi mediated reductions in cAMP concentration, across a variety of receptors, on standard automated fluorescence plate readers.

cADDis2 cAMP Indicator

  • Genetically encoded, single fluorescence emission
  • Packaged in a variety of viral vectors for use in any cell type
  • Decrease in fluorescence signal upon binding cAMP
  • Easy to use on standard fluorescence microscopes and automated fluorescence plate readers

Green fluorescent cADDis sensor in HEK293 cells responding to isoproterenol, a selective agonist for the endogenous β2 adrenergic receptor.

A New Way to Detect Gi Signaling

  • Simple, No cell lysis, No FRET, Fully reversible
  • Real-time detection of dynamic cAMP levels
  • All data obtained on Biotek Synergy MX plate reader

First ever direct readout of Gi mediated reductions in cAMP

  • Tuned expression of constitutively active Gαs raises basal levels of cAMP, without saturating cADDis
  • No pre-treatment with Gs agonists, Forskolin, or IBMX
  • Reveal the activity of Phosphodiesterases

Direct, Real-Time Gi Detection