cAMP is important as a second messenger in a variety of signaling pathways. The cAMP-Glo™ Max Assay was developed to test compounds for their ability to modulate cAMP levels in cells. This assay allows researchers to examine G-protein-coupled receptors (GPCRs) coupled with adenylate cyclase. A compound that activates or blocks GPCRs will alter intracellular cAMP levels, thus resulting in a signal change in the assay.
The cAMP-Glo™ Max Assay measures cAMP levels through protein kinase A (PKA), which is activated upon release of its regulatory subunits after binding to cAMP (Figure 1). PKA is a component of the cAMP Detection Solution, and once activated, PKA will use any ATP present to phosphorylate its substrate. Thus, the cAMP-Glo™ Max Assay produces a luminescent signal that is inversely proportional to the amount of cAMP present in the reaction. Increasing concentrations of cAMP leads to higher ATP turnover by PKA, resulting in less ATP to drive the luminescence reaction (using Ultra-Glo™ Recombinant Luciferase). Conversely, a sample with low amounts of cAMP will have high amounts of ATP and thus a high luminescent signal. In cell-based experiments where treatment results in changes to cAMP, it is important to understand that change in relation to potential cytotoxic effects or changes in cell number due to the treatment.
Figure 1. Schematic diagram of cAMP activation during the induction phase of the cAMP-Glo™ Max Assay (upper portion of the diagram). The shaded lower portion of the diagram depicts the assay after lysis with the cAMP Detection Solution plus the addition of the Kinase-Glo® Reagent (as described in Technical Manual #TM347).
The CellTox™ Green Cytotoxicity Assay detects the cytotoxic effects of compounds on cells. The CellTox™ Green Dye is a membrane impermeable, fluorescent, DNA-binding dye. It cannot penetrate viable cell membranes, but it can pass through the compromised membrane of dead cells to stain DNA (Figure 2). An increase in fluorescence occurs when the dye binds to DNA. The increased fluorescence correlates to increased cytotoxicity. Unlike other markers of necrotic cell death that require maintenance of enzymatic activity after release from the cytoplasm, staining of DNA provides a long lasting fluorescent signal.
The CellTox™ Green Dye is also stable to multiple fluorescence readings. This stable signal makes it ideal for extended treatment exposure times, for as long as 72 hours, allowing measurement of cytotoxicity either in real-time or as an endpoint assay. The reagent is nonlytic, allowing downstream assays to be multiplexed to gain more detailed data from each well.
Figure 2. CellTox™ Green Cytotoxicity Assay. CellTox™ Green Dye is excluded from viable cells, but it binds to DNA from dead cells with compromised membrane integrity.
If a decrease in cAMP levels is observed, it could be through inhibition of the adenylate cyclase pathway, an increase in cell death or an error in plating, with uneven cell distribution. Addition of CellTox™ Green Dye to the assay will help to differentiate these possible scenarios.
Here we describe multiplexing the CellTox™ Green Cytotoxicity Assay with the cAMP-Glo™ Max Assay to yield data on cAMP modulation as well as the cytotoxic effects of the compound being tested and the effects of cell plating variations. Furthermore, CellTox™ Green solution can be added directly to the cAMP-Glo™ Max Assay buffer preparation, simplifying experimental setup.
In this report we tested cAMP levels in cells in the presence of two compounds, forskolin and cholera toxin. We added CellTox™ Green Dye to these assays and determined if there were any effects on the luminescent signal produced by the cAMP-Glo™ Max Assay.
Materials to Be Supplied by the User
- IBMX (Acros Organics, Cat.# 228420010)
- Forskolin (Acros Organics, Cat.# 328240250)
- cAMP-Glo™ Max Assay (Promega, Cat.# V1681)
- CellTox™ Green Cytotoxicity Assay (Promega, Cat.# G8741)
- Cholera Toxin (from Vibrio cholera; Sigma-Aldrich, Cat.# C8052-1MG)
- Ro 20-1724 (Tocris Bioscience, Cat.# 0415)