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Markossian S, Grossman A, Arkin M, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-.
Tao Wang , Zhuyin Li , Mary Ellen Cvijic , Litao Zhang , and Chi Shing Sum .
Published November 20, 2017 .
Cyclic adenosine monophosphate (cAMP) is an important intracellular second messenger in GPCR signal transduction. Agonist activation of GPCRs that couple to the Gαs protein leads to an increased production of intracellular cAMP levels, whereas activation of GPCRs that couple to the Gαi protein leads to reduced production of intracellular cAMP levels. Both of these intracellular cAMP changes are mediated through the modulation of adenylate cyclase activity. cAMP regulates the activity of cAMP-dependent protein kinase A (PKA), which plays an important role in a variety of downstream cellular processes.
A number of reagent kits are available on the market that can be used to measure intracellular cAMP levels. These include the HTRF cAMP kit from Cisbio, the LANCE cAMP kit from PerkinElmer, the HitHunter cAMP kit from DiscoverX and the cAMP Direct Immunoassay Kit from Abcam and BioVision. These assays are all based on the use of antibodies that specifically recognize both intracellular cAMP and an exogenous labeled cAMP conjugate that acts as a competitor, followed by detection of the labeled cAMP conjugate by a variety of detection technologies, including fluorescence resonance energy transfer (FRET) or enzymatic reactions. In addition, the antibody-independent GloSensor cAMP assay from Promega employs semi-split luciferase, which reassembles when bound to cAMP. The advantages and disadvantages of the various cAMP assay technologies are given in Table 1. This chapter focuses on the Cisbio HTRF cAMP kit to illustrate the methodology and considerations for development of assays that measure intracellular cAMP. The assay development principles discussed here can be easily applied to similar cAMP measurement kits or various other detection methodologies.
Comparison of popular cAMP assay technologies
The Cisbio cAMP assay uses homogeneous time-resolved fluorescence technology (HTRF) to measure cAMP in a non-separation, high throughput format. These kits are based on a competitive immunoassay using Eu 3+ cryptate-labeled anti-cAMP antibody and d2-labeled cAMP (Figure 1). Detailed guidance on various immunoassay methods are described in the chapter “Immunoassay Methods” within this NIH Assay Guidance Manual (1). In the HTRF cAMP assay, binding of these two molecules will bring the long-lived fluorescent donor Eu 3+ cryptate and short-lived florescent acceptor d2 into close proximity, which facilitates fluorescence resonance energy transfer to occur between the donor and the acceptor. The long fluorescent lifetime of Eu 3+ cryptate allows the introduction of a time delay of approximately 50 to 150 microseconds between the system excitation and fluorescence emission measurement. Together with the large Stokes shift of the donor fluorophore (337 nm excitation to 620 nm emission), the Eu 3+ cryptate-based time-resolved fluorescent technology eliminates non-specific short-lived emissions (e.g. fluorescence from compounds or plastic plates) (2). Furthermore, the use of a ratiometric readout between donor emission at 620 nm and acceptor emission at 665 nm is advantageous, particularly for reducing well-to-well variations or plate edge effects that are often seen in homogeneous assay formats. Cisbio has several cAMP kits that differ in the affinity of the antibody for cAMP and are suitable for detection of intracellular cAMP levels over several relative concentration ranges.
Principles of the HTRF cAMP Assay. Anti-cAMP cryptate and d2-labeled cAMP are the two main components of the detection reagents. cAMP produced intracellularly is detected following cell lysis via competition between the intracellular cAMP and d2-labeled (more. )
The use of positive and negative controls is important to define the dynamic range of the assay within each plate and for proper quality control from plate to plate and run to run. The inclusion of one or more reference compounds for concentration-response analysis is valuable for assessing the incidence of assay drift. More details can be found in the AGM chapters HTS Assay Validation (3) and Assay Operations for SAR Support (4).
To establish the relationship between the actual cellular response and the assay readout, a cAMP standard curve should be included with each run as an assay control. The level of cAMP is measured indirectly through competition with a labeled cAMP competitor for binding to the cAMP antibody. The more cAMP produced, the lower the measured signal, and vice versa. The actual cAMP level is determined by using a standard curve in which the signals are measured using various known concentrations of cAMP. The cAMP standard curve is usually represented in a semi-log plot of Em665/Em615×10 4 versus cAMP concentration (Figure 2A). The measured signal ratio displays an inverse sigmoidal relationship with the log concentration of cAMP. The cAMP level from an assay reaction is determined via interpolation from the standard curve (1).
Illustration of the importance of estimating potency values using concentration-response curves expressed in cAMP levels instead of HTRF signal. Panel A. A typical cAMP standard curve. Panel B. Agonist concentration-response curves expressed in cAMP levels (more. )
During assay development, it is important that the assay conditions (e.g. cell density, forskolin concentration, and agonist stimulation) are optimized so that the measured signals fall within the linear range of the standard curve. Due to the sigmoidal relationship between the signal and the cAMP level, signals at the top region of the cAMP standard curve can be too sensitive to changes in cAMP level, whereas signals at the bottom region can be too insensitive to the changes.
Figure 2 illustrates the importance of converting the measured signals to cAMP levels to accurately determine the potency values from concentration-response curves. Figure 2A shows a typical cAMP standard curve. Figure 2B is a series of concentration response curves simulated to closely resemble actual data from the response characteristics of an agonist that mediates inhibition of forskolin-stimulated cAMP production in a Gαi-coupled receptor. Increasing the concentration of the agonist causes a reduction in intracellular cAMP levels. Each of the nine curves shown in the panel represents different dilutions from the original lysate from 1X to 0.04X dilutions. Since the curves are simply generated by dilution of the same lysates, the agonist EC50 values are the same for all the curves (i.e., 100 nM). For illustration purposes, if these agonist curves are converted back to HTRF signal in accordance with the standard curve, another series of curves is generated as shown in Figure 2C. The estimated EC50 values from Figure 2C erroneously increase over a 4-fold range but none of these values accurately reflect the actual EC50 value. This phenomenon is a result of the semi-logarithmic relationship between the measured signal and cAMP level. Similar discussion on the importance of using a cAMP standard curve to convert measured signal ratios to levels of cAMP and how basing the activity measurement on HTRF ratio may skew the potency and efficacy estimation are also described in the literature (5,7). Furthermore, the use of a calibration curve and the selection of an appropriate model is thoroughly discussed in the AGM chapter Immunoassay Methods (1).
As indicated in the above flowchart, assay development should start from selecting the appropriate endogenously expressing cells or transfecting cells to overexpress the target receptor. If cell lines stably expressing the receptors are generated, multiple clones can be selected initially either by FACS to assess the surface protein expression or by qPCR for gene expression. The level of receptor expressed on the cell surface impacts both the potency and efficacy of agonists. Figure 3 illustrates how the estimated potency values increase for a set of agonists as the expression level of target receptor increases. Furthermore, a partial agonist may behave as a full agonist due to a high level of receptor expression, which leads to high receptor reserve. During the early lead discovery phase, it may be advantageous to utilize cell lines of high receptor expression to enhance the sensitivity of detecting hit compounds. Nevertheless, it would be important to assess multiple cell clones expressing receptors at various levels if correlation with other assay readouts is desirable in the lead optimization phase.
Dependence of agonist potency (EC50 values) on the level of receptor expression. Shown are individual compound potency values that were measured in cells lines expressing a given level of receptor. Different compounds are shown in symbols and lines of (more. )
Once a cellular model is selected or generated, for a Gαs-coupled receptor, assay development may begin by determining the concentration-response relationship of a known agonist at various cell densities. Figure 4 shows an example of such agonist and cell density titration curves. As shown in the figure, it is important to note that when there are too many cells, the signal-to-background may be adversely affected. The agonist concentration-response curves are examined to identify concentrations in which the response falls within the linear detection range of the cAMP standard curve. A cell density that produces the best dynamic range and expected EC50 value of the agonist is selected.
Examples of (A) agonist-concentration response curves at various cell densities for a Gαs-coupled receptor and (B) agonist-concentration response curves expressed in cAMP level. In this example, when the cell density is too high as with 6 k/cells, (more. )
For a Gαi-coupled receptor, its activation needs to be assessed under a detectable cAMP level. This can be achieved using forskolin or a known agonist of a Gαs-coupled receptor to stimulate cAMP production, as shown in Figure 5. Similarly, a combination of appropriate forskolin concentration and cell density that can stimulate cAMP production to a level that is within the linear detection range and generate optimal assay dynamic range would be selected. Subsequently, the agonist response can be assessed at the chosen conditions to ensure its potency agrees with anticipated values.
Examples of (A) forskolin concentration response-curves at various cell densities for a Gαi-coupled receptor, (B) an agonist concentration-response assayed at 3 μM forskolin and 1,500 cells/well as selected from Panel A, and (C) the agonist (more. )
For either Gαs- or Gαi-coupled receptors, following the determination of agonist response, the potency of antagonist can be assayed at a selected agonist concentration between EC50 to EC80. A summary of assay optimization and troubleshooting guidelines is given in Table 2.
Key assay optimization parameters and troubleshooting guidelines
The following guidelines were developed for assays using suspension cells for high throughput mode; however, the assay can be adapted easily for adherent cells. Volumes are given for a 384-well assay (20 μL assay reaction) and can be adjusted proportionally to other plate densities.
The activation of adelnylate cyclase by Gαs, in the presence of an agonist, the generation of intracellular cAMP and its measurement by HTRF technology is shown in Figure 6.
cAMP Measurement for Agonists of a Gαs-Coupled Receptor. Activation of the receptor by an agonist causes an exchange of GDP for GTP in the G protein complex that subsequently dissociates into a Gβγ dimer and a Gαs monomer. (more. )
Step 1: Grow cells in tissue culture flasks for 1-3 days in the corresponding cell culture medium.
Select a cell line containing the target GPCR of interest.A negative control cell line as a counter screen may either be the parental cell line devoid of the target or a cell line expressing an unrelated GPCR that couples to the same G protein.
Make sure the cells are healthy and active in the log phase of growth and are maintained in the same way in all experiments, as the state of the cells may affect the receptor response.
Limit cell passage in concordance with assay performance statistics. Ideally, to avoid variation in cellular response due to changes in the state of the cells or receptor expression, expand the cell culture to the quantities required for the entire screening campaign in one large batch of a single passage and prepare cryopreserved cells as one batch. Various manufacturers of cell culture reagents offer different cryopreservation reagents and methods. During screening, recover the cells and use them as needed.
Step 2: Dissociate the cells from the flask and centrifuge at 1000 rpm for 5 minutes.
Use of Trypsin or other enzymatic/non-enzymatic cell dissociation agents should be tested prior to screening.
Some receptor proteins may tolerate trypsinization, but other enzymatic reagents such as TrypLE (Thermo) or non-enzymatic reagents such as CellStripper (Corning) may be used if needed.
Step 3: Aspirate the medium and re-suspend the cells in assay buffer. Count and dilute the cells to the proper densities in assay buffer.
In most cases, HBSS or Dulbecco’s PBS (DPBS) buffers can be used: 1X HBSS/20mM HEPES with or without 0.1% BSA (fat acid-free) or DPBS with Ca 2+/ Mg 2+ with or without 0.1% BSA.
Cell densities should be determined during assay development in cell titration experiments such that the measured signal falls within the linear range of the cAMP standard curve while yielding the best assay window determined with a reference agonist.
Using too many cells, and hence the presence of too many receptors, may reduce the effective free concentrations of ligands and cause the assay to bottom-out and therefore limit the ability to differentiate potent compounds. Too many cells may also saturate the cAMP assay reagents.
Step 4: Add 3-isobutyl-1-methylxanthine (IBMX) or other phosphodiesterase inhibitor to the cell suspension. IBMX is a competitive nonselective phosphodiesterase inhibitor which inhibits the degradation intracellular cAMP. It is generally used at a final concentration of 0.1 mM in the assay, but the impact of phosphodiesterase inhibitor on compound potency and the required concentration should be determined according to the assays and cell lines being used. Dispense 10 μL of cells to assay plates pre-dotted with compounds. (Note that depending on the extent of activity of phosphodiesterase in the cells, the use of inhibitor is optional.)
IBMX can be made at high concentration (0.5 M) and aliquoted and stored at -20 ° C to avoid multiple freeze-thaw cycles.
Different assay plates may be tested. Please refer to Cisbio website for microplate recommendations (http://www.cisbio.com/usa/drug-discovery/htrf-microplate-recommendations).
The cell suspension is best prepared only when it is ready for dispensing to avoid deterioration of cellular response. In a large screening campaign, longevity of the cell suspension should be determined from the concentration-response curves.
Compounds dissolved in DMSO can be pre-dotted into assay plates in sub-microliter volume using an acoustic dispenser, such as Labcyte Echo or EDC Biosytems ATS, or pre-diluted in an assay buffer prior to addition. Tolerance of the cellular response to DMSO should be investigated during assay development. Most cells can tolerate up to 1% DMSO in the assay. An example of a DMSO tolerance test is shown in Figure 7.
Cells and other reagents can be dispensed by various peristaltic pump-based liquid handlers, such as Thermo Combi-drop or the BioTek Washer Dispenser, or any of the widely available tip-based liquid handlers.
An example of a DMSO tolerance test carried out by conducting the cAMP assay in the presence of different concentrations of DMSO. Various parameters are assessed to identify the acceptable range of DMSO without adversely affecting the performance of the (more. )
Step 5: Cover the assay plate and incubate for 30 minutes at room temperature (RT).
The duration of assay incubation depends on the kinetic properties of the compounds. Potency values can be underestimated if the incubation is insufficient to achieve steady state conditions.
Step 6: Prepare the cAMP standard curve according to the manufacturer’s instructions.
Step 7: Dispense 5 μL/well of diluted d2-labeled cAMP conjugate followed by 5 μL/well of cryptate-labeled anti-cAMP antibody to the cell plate and standard curve plate. Both MultiDrop and Tempest liquid handling instruments are compatible for HTRF reagents.
d2-labeled cAMP conjugate and cryptate-labeled anti-cAMP antibody are reconstituted per manufacturer’s instructions.
Working solutions are prepared by diluting the concentrated stock 20-fold using lysis buffer provided in the kit.
Step 8: The plates are incubated for 1 hour at RT and read using instruments such as a PerkinElmer Envision with a protocol that is set and optimized for HTRF detection.
Fluorescence intensity is measured at emission of 665 nm and 615 nm (Em665 and Em615), with excitation at 350 nm.
Plates can be read repeatedly to ensure that the antibody binding reaches steady state.Step 9: Convert the results from the reader into cAMP levels using the cAMP standard curve.
Readings are generally expressed as Em665/Em615×10 4 , but may vary depending on the exact detection instrument being used.
Step 10: Data analysis. See Figure 8.
An example of an agonist concentration-response curve in Gαs-coupled receptors. Panel A. The agonist response is plotted as the HTRF ratio (Em665/Em615×10 4 ) along with the cAMP standard curve. Note that the agonist produces signals that (more. )
In this format, an antagonist displaces an agonist for a Gαs coupled receptor and the resulting inhibition of adenylate cyclase is measured using the HTRF assays, as shown in Figure 9.
cAMP Measurement for Antagonists of a Gαs-Coupled Receptor. An antagonist prevents the activation of the receptor by an agonist which prevents G protein complex dissociation. The adenylate cyclase is not activated and the generation of intracellular (more. )
The procedure for determination of antagonist activity of a Gαs-coupled receptor using cAMP measurement is essentially the same as the procedure described above for detection of agonist activity. In the antagonist format, an agonist is added at concentrations that trigger 50-80% of the maximum response (EC50-80) of the agonist. The rationale and precautions for each step can be referred to in the above agonist assay protocol, except for those specific to antagonist assays. All compounds of interest should also be tested in the agonist mode to ensure the absence of agonist activities.
Step 1: Grow cells in flask for 1-3 days in the corresponding cell culture medium.
Step 2: Dissociate the cells from the flask and centrifuge at 1000 rpm for 5 minutes.
Step 3: Aspirate the medium and re-suspend the cells in assay buffer. Count and dilute the cells to the desired concentration in assay buffer.
The working concentration of the cell suspension is twice as much as in agonist mode due to the volume difference. For example, if 1×10 6 cell/ml is used in the agonist assay, then 2×10 6 cells/ml is prepared for the antagonist assay.
Step 4: Add IBMX, which is generally used at a final concentration of 0.1 mM in the assay, but the impact of phosphodiesterase inhibitor on compound potency and the required concentration should be determined according to the assays and cell lines being used. Dispense 5 μL of cells into assay plates that have been pre-dotted with test compound in DMSO or pre-diluted in an assay buffer. (Note that depending on the extent of activity of phosphodiesterase in the cells, the use of inhibitor is optional.)
Step 5: Cover the plate and incubate for 15-30 minutes at room temperature (RT).
Depending on the kinetic properties of test compounds relative to the agonist used for stimulation, this step can be adjusted from no incubation to an extended incubation time as needed.
Step 6: Add 5 μL of the agonist solution diluted in assay buffer. Incubate the plates at RT for 30 minutes.
The final concentration of the agonist chosen should result in 50 to 80% of the maximum response of the agonist, as determined during development of the agonist assay.
The working concentration is prepared at 2X the desired final concentration.At the chosen EC50-80 concentration of agonist, the measured agonist response should not exceed the linear range of the cAMP standard curve.
Step 7: Prepare the standard curve according to the manufacturer’s instructions.
Step 8: Dispense 5 μL of diluted d2-labeled cAMP conjugate followed by 5 μL of cryptate-labeled anti-cAMP antibody into the cell plate and the standard curve plate.
Step 9: Incubate the plates for 1 hour at RT and measure the signal with a PerkinElmer Envision reader (or equivalent) using a protocol that is optimized for HTRF measurements.
Step 10: Convert the results from the reader into cAMP levels using the cAMP standard curve.
Readings are generally expressed as Em665/Em615×10 4 .Step 11: Data analysis. See Figure 10.
An example of antagonist concentration-response curves in Gαs-coupled receptors. Panel A. The antagonist response is plotted as the HTRF ratio (Em665/Em615×10 4 ) along with the cAMP standard curve. Note that the antagonist produces signals (more. )
The following guidelines are developed for assays carried out using suspension cells for high-throughput mode, although the assay can be adapted easily for adherent cells. Volumes are given for a 384-well assay (20 μL assay reactions) and can be adjusted proportionally to other plate density formats.
In assaying a Gαi -coupled receptor, forskolin is typically used to increase the level of cAMP so that the lowering of the cAMP level due to receptor negative regulation of adenylate cyclase can be observed (Figure 11). Alternatively, the cells can be stimulated to produce cAMP by an agonist to a Gαs-coupled receptor that is also present in the cells.
cAMP Measurement for Agonists of a Gαi-Coupled Receptor. Treatment of cells with forskolin results in an increase in cellular cAMP levels. The addition of an agonist decouples the G protein complex that subsequently dissociates into a Gβγ (more. )
Step 1: Grow cells in tissue culture flasks for 1-3 days in the corresponding cell culture medium.
Select a cell line containing the target GPCR of interest.A negative control cell line as a counter screen may either be the parental cell line devoid of the target or a cell line expressing an unrelated GPCR that couples to the same G protein.
Make sure the cells are healthy and active in the log phase of growth and are maintained in the same way in all experiments, as the state of the cells may affect the receptor response.
Control cell passage number based on assay performance. Ideally, to avoid variation in cellular response due to changes in the state of the cells or receptor expression, expand the cell culture to the quantities required for the entire screening campaign in one large batch of a single passage and prepare cryopreserved cells as one batch. Various manufacturers of cell culture reagents offer different cryopreservation reagents and methods. During screening, recover the cells and use them as needed.
Step 2: Dissociate the cells from the flask and centrifuge at 1000 rpm for 5 minutes.
Use of Trypsin or other enzymatic/non-enzymatic cell dissociation agents should be tested prior to screening.
Some receptor proteins may tolerate trypsinization, but other enzymatic reagents such as TrypLE (Thermo) or non-enzymatic reagents such as CellStripper (Corning) may be used if needed.
Step 3: Aspirate the cell culture medium and re-suspend the cells in assay buffer. Count and dilute the cells to the proper densities in assay buffer.
In most cases, HBSS or Dulbecco’s PBS (DPBS) buffers can be used: 1X HBSS/20mM HEPES with or without 0.1% BSA (fat acid-free) or DPBS with Ca 2+/ Mg 2+ with or without 0.1% BSA.
Cell densities should be determined during assay development in cell titration experiments such that the measured signal falls within the linear range of the cAMP standard curve while yielding the best assay window.
Using too many cells, and hence the presence of too many receptors, may reduce the effective free concentrations of ligands and cause the assay to bottom-out and therefore limit the ability to differentiate potent compounds. Too many cells may also saturate the cAMP assay reagents.
Step 4: Prepare a solution containing the desired amount of forskolin (to stimulate cAMP production so that the inhibitory effect of agonist on the cAMP level can be detected.) Add IBMX, generally at a final concentration of 0.1 mM, or other phosphodiesterase inhibitor and dispense 5 μL of the mixture into assay plates pre-dotted with compounds, followed by dispensing 5 μL of cell suspension. (Depending on the extent of activity of phosphodiesterase in the cells, the use of an inhibitor is optional. The impact of a phosphodiesterase inhibitor on compound potency and the required concentration should be determined according to the assays and cell lines being used.)
Forskolin can be prepared as a 10 mM stock in DMSO in a glass vial and kept at RT.The concentration of forskolin to be used in the assay should be optimized to ensure that the measured signal falls within the linear range of the cAMP standard curve while yielding the best assay window. Cell density and forskolin concentration can be optimized simultaneously by a three-way titration that includes cells and forskolin at different concentrations in the absence and presence of a reference agonist.
IBMX can be made at high concentration (0.5 M), aliquoted and stored at -20 ° C to avoid multiple freeze-thaw cycles.
Different assay plates may be tested. Please refer to the Cisbio website for microplate recommendations (http://www.cisbio.com/usa/drug-discovery/htrf-microplate-recommendations).
The cell suspension is best prepared only when it is ready for dispensing to avoid deterioration of cellular response. In a large screening campaign, longevity of the cell suspension should be determined from the concentration-response curves.
Compounds dissolved in DMSO can be pre-dotted into assay plates in sub-microliter volume using an acoustic dispenser, such as Labcyte Echo or EDC Biosystem ATS, or pre-diluted in an assay buffer prior to addition. Tolerance of the cellular response to DMSO should be investigated during assay development. We found that most cells can tolerate up to 1% DMSO in the assay.
Step 5: Cover the assay plate and incubate for 30 minutes at room temperature (RT).
The duration of assay incubation depends on the kinetic properties of the compounds. Potency values can be underestimated if the incubation is insufficient to achieve equilibrium.
Step 6: Prepare the cAMP standard curve according to the manufacturer’s instructions.
Step 7: Dispense 5 μL/well of diluted d2-labeled cAMP conjugate followed by 5 μL/well of cryptate-labeled anti-cAMP antibody to the cell plate and cAMP standard curve plate.
d2-labeled cAMP conjugate and cryptate-labeled anti-cAMP antibody are reconstituted per manufacturer’s instructions.
Working solutions are prepared by diluting the concentrated stock 20-fold using lysis buffer provided in the kit.
Step 8: The plates are incubated for about 1 hour at RT and read using an instrument such as the PerkinElmer Envision with a protocol that is set and optimized for HTRF detection.
Fluorescence intensity is measured at Em665 and Em615, with excitation at 350 nm. Plates can be read repeatedly to ensure that the antibody binding reaches steady state.Step 9: Convert the results from the reader into cAMP levels using the cAMP standard curve.
Readings are generally expressed as Em665/Em615×10 4 .Step 10: Data analysis. See Figure 12.
An example of agonist concentration-response curves in Gαi-coupled receptors. Panel A. The agonist response is plotted as the HTRF ratio (Em665/Em615×10 4 ) along with the cAMP standard curve. Note that the agonist produces signals that (more. )
In this format, an antagonist displaces an agonist for a Gαi coupled receptor and the resulting inhibition of adenylate cyclase is measured using the HTRF assays, as shown in Figure 13.
cAMP Measurement for Antagonists of a Gαi-Coupled Receptor. Treatment of cells with forskolin results in an increase in cellular cAMP levels. The addition of an agonist decouples the G protein complex that subsequently dissociates into a Gβγ (more. )
The procedure for determination of antagonist activity of a Gαi-coupled receptor using cAMP measurement is essentially the same as the procedure described above for detection of agonist activity. In the antagonist format, an agonist is added at concentrations that trigger 50-80% of the maximum response (EC50-80) of the agonist. The rationale and precautions for each step can be referred to in the above agonist assay protocol, except for those specific to antagonist assays. All compounds of interest should also be tested in the agonist mode to ensure the absence of agonist activities.
Step 1: Grow cells in flask for 1-3 days in the corresponding cell culture medium.
Step 2: Dissociate the cells from the flask and centrifuge at 1000 rpm for 5 minutes.
Step 3: Aspirate the medium and re-suspend the cells in assay buffer. Count and dilute the cells to the desired concentration in assay buffer. Dispense 5 μL of cells into assay plates that have been pre-dotted with compounds in DMSO or pre-diluted in an assay buffer. Incubate the plate at RT for 15-30 minutes.
Depending on the kinetic properties of the test compounds relative to the agonist used for stimulation, this step can be adjusted from no incubation to an extended incubation time as needed.
Step 4: Prepare a solution containing the desired concentration of forskolin, IBMX (generally at a final concentration of 0.1 mM) and agonist, and dispense 5 μL of the mixture into the assay plates pre-dotted with compounds. (Depending on the extent of activity of phosphodiesterase in the cells, the use of inhibitor is optional. The impact of phosphodiesterase inhibitor on compound potency and the required concentration should be determined according to the assays and cell lines being used.)
The final concentration of the agonist chosen should result in 50 to 80% of the maximum response of the agonist, as determined during development of the agonist assay.
The working concentration of forskolin, IBMX and agonist solution mixture is prepared at 2X the desired final assay concentration.
At the chosen EC50-80 concentration of agonist, the measured agonist response should not exceed the linear range of the cAMP standard curve.
Step 5: Cover the plate and incubate for 30 minutes at room temperature (RT).
Step 6: Prepare the standard curve according to the manufacturer’s instructions.
Step 7: Dispense 5 μL diluted d2-labeled cAMP conjugate followed by 5 μL of cryptate-labeled anti-cAMP antibody into the cell plate and the standard curve plate.
Step 8: Incubate the plates for 1 hour at RT and measure the signal with a PerkinElmer Envision using a protocol that is optimized for HTRF detection.
Step 9: Convert the results from the reader into cAMP levels using the cAMP standard curve.
Readings are generally expressed as Em665/Em615×10 4 .Step 10: Data analysis. See Figure 14.
Examples of agonist and antagonist concentration-response curves in Gαi-coupled receptors. Panel A. The agonist and antagonist responses are plotted as the HTRF ratio (Em665/Em615×10 4 ) along with the cAMP standard curve. Note that both (more. )
The authors would like to thank Nathan Cheadle, Jie Pan and Melissa Yarde for the review of this guidance.
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