May 1, 2016 (Vol. 36, No. 9)
Optimization Process of a TR-FRET cAMP Assay
G-protein coupled receptors (GPCRs) are a major class of targets for drug discovery. Cyclic AMP (cyclic adenosine 3′,5′-monophosphate) is a second messenger and one of the most important intracellular mediators. Changes in intracellular cAMP levels correlate with GPCR activation. Measurement of cAMP levels therefore offer an assay for GPCR high-throughput screening.
Cells are stimulated to either increase or decrease intracellular cAMP levels. For Gαi-coupled receptors, an elevation in intracellular cAMP can be stimulated using forskolin. After stimulation, HTRF® Technology (Homogenous Time Resolved Fluorescence) can be used for the direct quantitative determination of cAMP.
The Cisbio HiRange cAMP kit is based on a competitive immunoassay between native cAMP produced by cells and the cAMP labeled with the dye d2. The specific signal (i.e., energy transfer signal) is inversely proportional to the concentration of native cAMP in the standard or sample. Compared to other HTRF cAMP assays, the HiRange kit has an extended signal-to-background ratio. The kit allows the measurement of agonist and antagonist effects on Gαs– and Gαi-coupled receptors in different cell lines.
In order to optimize the assay, a suitable number of cells needs to be used per well, and the optimum cell density is the number of cells that produce the highest amplitude between the inactivated and activated cellular states.
Cell Density and Dynamic Range
In a study, three different cell concentrations (1,000, 3,000, and 10,000 cells per well) were tested. Two separate assays were made and in the first one, the standard curve was prepared to see the basic functionality of the system. The second assay was used to perform the cell number optimization.
The standard curve assay was performed according to kit instructions using standards, blank and control, assayed in six replicates.
The CHO-1 cells were grown in suspension, and plated as 1,000, 3,000, or 10,000 cells per well, and activated with different concentrations of forskolin. The phosphodiesterase inhibitor IBMX was added to the compound buffer to prevent degradation of cAMP. The cell assay was performed according to kit instructions. All samples were assayed in triplicate.
The Thermo Scientific™ Varioskan™ LUX multimode reader, controlled with Thermo Scientific SkanIt™ software, was used to measure the plates by the parameters listed in Table 1. Using a special autorange feature that automatically selects the optimal measurement gain for each sample, the instrument provides high sensitivity and wide dynamic range for every run without the need for manual adjustments. This allows the user to measure low and high signal samples reliably within the same assay and achieve consistent results.
For all HTRF assays, the result is expressed as the ratio of the acceptor wavelength (665 nm) and the donor wavelength (620 nm), in order to eliminate interferences. Delta F (%) was determined for all samples from ratio values according to kit instructions.
Examination of the assay dynamic range after stimulation of cAMP with forskolin reveals differences based on the number of cells per well. According to the results, 10,000 cells per well provided the widest dynamic range in this study (Figure 1).
The standard curve was created with SkanIt software from the calculated Delta F values with four parameter logistics as the fitting method (Figure 2). The standard curve shows a very wide dynamic range, and the calculated effective dose (ED50) value, 24 nM, correlates well with the reported value in the kit documentation.
In addition to the HTRF calculations, the Z prime value was also calculated for the 10,000 cell assay, to provide a dimensional parameter to describe assay quality. Values above 0.5 are considered very good. A value of 0.94 was achieved with the system.
The optimization process of the HTRF cAMP assay was very straightforward. In addition to excellent performance, the flexibility of measurement setup and ease of use of the Varioskan LUX multimode microplate reader are valuable features for assay development and screening. By using the automatic dynamic range functionality of the reader, the measurement settings are always optimal, making screening assays both easy and robust.