GloSensor™ Technology: Intracellular Biosensors for cAMP, cGMP, and Protease Detection

GloSensor™ technology provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells with sensitivity, linearity and specificity.

GloSensor™ luciferase is circularly permuted, joining the wild-type N- and C-termini with a polypeptide or protein domain and creating engineered termini elsewhere within the structure. Analyte binding or protease cleavage leads to a conformational shift in the biosensor that promotes large increases in luminescence activity. The magnitude of the luminescence increase is directly proportional to the amount of analyte or protease activity present.

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Simple, Scaleable, Live-Cell Protocol

The live-cell, non-lytic assay protocol is easy to perform, allowing biosensor activity to be monitored continuously or at a single time point. The kinetic profile of cell signaling events can be followed in real time and the assays easily scale to high-density microplate formats, including 96-, 384- and 1536-well plates.

Easy, scalable, robust assay:

  • Pre-equilibrate cells with GloSensor™ cAMP Reagent for ~2 hours.
  • Treat with specific agonists/antagonists or library compounds.
  • Measure luminescence after 10–30 minutes either continuously or at a single time point.

GloSensor™ cAMP Assay: Monitor Changes in Intracellular cAMP in a Live-Cell Assay

The GloSensor™ cAMP Assay can be used to monitor activation of endogenous or overexpressed Gs- or Gi-coupled 7-TM, G-protein coupled receptors (GPCRs), together with phosphodiesterases (PDEs) that are responsible for degrading intracellular cAMP. Intracellular changes in cAMP concentration can be followed using a live-cell, non-lytic assay format, allowing real-time monitoring of changes in cAMP concentration.

Different biosensors are available for use in the GloSensor™ cAMP Assay. In each case, a cAMP binding domain is fused to a circularly permuted form of firefly luciferase. The sequence for each differs, giving biosensors with a range of affinities for cAMP. In general, the pGloSensor™-22F cAMP Plasmid is best-suited for most applications, providing a unique combination of sensitivity and dynamic range. The 22F construct is sensitive enough to monitor inverse agonist activity or Gi-coupled receptor activation in the absence of added forskolin, but possess a large enough dynamic range to monitor very large increases in intracellular cAMP concentration.

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Live-Cell, Non-Lytic Assay Format Allows Kinetic or Endpoint Measurements of cAMP

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Kinetic response of GloSensor™ cAMP compared to a cAMP immunoassay. HEK293 cells transiently expressing the pGloSensor™-22F cAMP Plasmid were treated sequentially with 1 µM isoproterenol (ISO), 10 µM propranolol (PRO), and 10 µM forskolin (FSK). The live-cell, non-lytic GloSensor™ cAMP Assay protocol was used to monitor cAMP levels, whereas replicate wells were lysed at each time point for the cAMP immunoassay (Enzo Life Sciences). Samples above approximately 20 nM required dilution for the cAMP immunoassay to be within the linear range of the standard curve. (ISO = ADRB2 agonist, PRO = ADRB2 antagonist, FSK = cell permeable activator of adenylyl cyclase).

Quantitative Endpoint Analysis of cAMP Levels

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Endpoint assay to detect cAMP. HEK293 cells were transiently transfected with pGloSensor™-22F cAMP Plasmid and treated with the compounds indicated. Luminescence was measured 10 minutes after addition of varying concentrations of the respective compounds, and this value was divided by a pre-read measurement taken prior to compound delivery to determine fold response.

GloSensor™ cGMP Assay: Monitor Changes in Intracellular cGMP in a Live-Cell Assay

The GloSensor™ cGMP Assay is used to monitor changes in the intracellular concentration of the second messenger cGMP. The assay can be used to monitor activation of endogenous or overexpressed guanylyl cyclases, including soluble guanylyl cyclase (sGC) or atrial natriuretic peptide receptor (NPRA and NPRB) isoforms, together with the phosphodiesterases (PDEs) that are responsible for degrading intracellular cGMP. Intracellular changes in cGMP concentration can be followed using a live-cell, non-lytic assay format, allowing real-time monitoring of changes in cGMP concentration.

Different biosensors are available for use in the GloSensor™ cGMP Assay. In each case, a cGMP binding domain is fused to a circularly permuted form of firefly luciferase. The cGMP binding domain and sequence for each differs, giving biosensors with different affinities for cGMP. In general, the 40F construct is preferred when overexpressing a guanylyl cyclase, providing maximal dynamic range at high intracellular levels of cGMP. In contrast, the 42F construct is preferred when assaying an endogenous guanylyl cyclase, providing maximal sensitivity for lower intracellular levels of cGMP.

GloSensor™ cGMP vectors and stable cell lines are available as custom assay material.

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Monitor Changing cGMP Levels in a Kinetic, Real-Time Assay

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Real-time cGMP monitoring. HEK293 cells transiently expressing the 42F construct were treated with varying doses of sodium nitroprusside (SNP), a nitric oxide donor, to activate the endogenous soluble guanylyl cyclase (sGC). The live-cell, non-lytic GloSensor™ cAMP Assay protocol was used to monitor the cGMP response in real-time.

GloSensor™ Protease Assay: Luminescent Detection of Protease Activity

The GloSensor™ Protease Assay is used to monitor protease activity both in vitro and in vivo. The approach utilizes circularly permuted forms of luciferase where the wild-type N- and C-termini are tethered with a peptide containing a protease cleavage site. Once cleaved by the corresponding protease, a conformational constraint is lifted, resulting in large increases in luminescence activity. Peptides containing protease cleavage sites up to 20 amino acids in length have been validated in vitro, using this technology, including proteases that require P’ residues for efficient cleavage.

A novel GloSensor™ protease construct, the 30F plasmid, was developed for live-cell, non-lytic assays of protease activity for use in both cell culture and in vivo models. When engineered with a caspase-3/7 cleavage site, the GloSensor™ caspase 3/7 biosensor enables a sensitive, real-time approach for the detection of caspase-3/7 activity enabling a method for in vivo measure of apoptosis in animal models. The GloSensor™ caspase 3/7 biosensor, together with a similar construct designed to detect granzyme B, have also been used for the detection of target cell apoptosis mediated by in vitro-derived or ex vivo cytotoxic T lymphocytes (CTLs).

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The GloSensor™ caspase 3/7 biosensor plasmid, stable cells lines and granzyme biosensor plasmids are available as custom assay materials. These biosensor plasmids can be used directly or modified by the insertion of novel cleavage sites to create biosensors for a particular protease of interest.

  

Live Cell Protocol

The protocol for live-cell, non-lytic assays using the GloSensor™ protease assay is identical to that of the GloSensor™ cAMP Assay, and uses the GloSensor™ cAMP Reagent as a substrate. The GloSensor™ cAMP Reagent provides the substrate needed for biosensor catalysis and its formulation has been extensively validated for use in this application. For in vivo assays, Vivo-Glo™ Luciferin is used as the GloSensor™ substrate.