increasing the flexibility of the lance camp detection kit
TRANSCRIPT
Journal of Pharmacological and Toxicological Methods 71 (2015) 42–45
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Journal of Pharmacological and Toxicological Methods
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Brief communication
Increasing the flexibility of the LANCE cAMP detection kit
Morag Rose Hunter, Michelle Glass ⁎Department of Pharmacology & Clinical Pharmacology, University of Auckland, Private Bag 92019, Auckland, New Zealand
Abbreviations:cAMP,cyclicadenosinemonophosphate;GTR-FRET, time-resolvedfluorescence resonance energy transf⁎ Corresponding author. Tel.:+64 9 9236247.
E-mail addresses: [email protected] (M.R. Hun(M. Glass).
http://dx.doi.org/10.1016/j.vascn.2014.10.0081056-8719/© 2014 Elsevier Inc. All rights reserved.
a b s t r a c t
a r t i c l e i n f oArticle history:Received 18 August 2014Accepted 26 October 2014Available online 3 December 2014
Keywords:cAMPAdenylyl cyclaseHigh throughput screeningCell signalling assaysG-protein coupled receptors
Introduction: The detection of cAMP signalling is a common endpoint in the study of G-protein coupled re-ceptors. A number of commercially available kits enable easy detection of cAMP. These kits are based on compe-tition for a cAMP binding site on an antibody or cAMP binding protein and as such have a limited dynamic range.Here,we describe the optimisation of the commercially-available LANCE cAMPdetection kit (PerkinElmer) to en-able detection in cell lysates. This kit has been designed for use with live cells, with detection reagents applied tocells withoutwash steps. The standard protocol therefore requires that all assay reagents are compatiblewith theantibody and thefinal fluorescent detection stage, limiting the range of assaymedia and test compounds that canbe utilised. The entire experiment must be repeated if cAMP levels fall outside the limited dynamic range. Herewe describe a modified protocol that enables the assay to be performed on cell lysates, thereby overcomingthese limitations. Methods: In this modified protocol, cells are stimulated for a cAMP response in standardmedia/buffers, washed and then lysed. The cell lysate is then assayed using a modified protocol for the LANCEcAMP detection kit. Samples were tested for stability following a freeze–thaw cycle. Results: The modifiedLANCE cAMP detection protocol gives a reproducible measurement of cAMP in cell lysate. Lysate samples remainstable when stored at−80 °C. Discussion: Separating the stimulation and detection phases of this cAMP assayallows a vast array of cell stimulation conditions to be tested. The lysate-modified protocol for the LANCEcAMP detection kit therefore increases the flexibility, versatility and convenience of the assay. As samples are in-sensitive to freeze–thaw, it enables retesting of samples under different dilution conditions to ensure that allsamples remain within the dynamic range of the standard curve.
© 2014 Elsevier Inc. All rights reserved.
1. Introduction
The detection of drug-mediated alterations in cellular cAMP concen-trations is a commonmethod of drug discovery. Intracellular cAMP levelsare tightly regulated by adenylyl cyclase enzymes that convert adenosinetriphosphate to cAMP, as well as by phosphodiesterases that catalysecAMP degradation. Isoforms of adenylyl cyclase are activated or inhibitedvia direct interactionwithG-protein subunits (stimulated byGs, inhibitedby Gi/o, and therefore under the control of G-protein coupled receptors(GPCRs)), or by changes in intracellular Ca2+ and calmodulin. cAMP inturn regulates a range of cAMP-dependent protein kinases, resulting inthe phosphorylation of protein targets (see Hofer, 2012 for a recentreview).
The last decade has seen a surge in newmethodologies for the detec-tion of intracellular cAMP (see Hill, Williams, & May, 2010 for review),with an increasing emphasis onmethods adaptable for high throughputscreening for drug discovery. Many of these methods are now available
PCR,G-proteincoupledreceptor;er.
ter), [email protected]
in the form of convenient “kits”. One such kit is the LANCE cAMPkit (PerkinElmer). The LANCE cAMP assay is a time-resolved fluores-cence resonance energy transfer (TR-FRET) immunoassay, in whichbiotinylated-cAMP and europium-W8044 chelate-labelled streptavidincompete for cAMP binding sites on an Alexa-Fluor 647-labelled anti-body. A light pulse at 340 nm then excites the europium-chelate mole-cule of the tracer, leading to energy transfer to the Alexa fluorophore,which in turn emits light at 665 nm. Thus, the greater the level ofcAMP in the test sample, the higher the level of competition and thelower the signal emitted. Using the manufacturer's protocol for theLANCE cAMP kit, detection reagents are applied to live cells suspendedin a minimal assay buffer, and drug stimulation is performed at roomtemperature for an extended period of time. The fluorescent readoutis then converted to a concentration of cAMP per assay point, utilisinga cAMP standard curve constructed from pure cAMP and the same de-tection reagents.
As for all such assays, the validity of the data is dependent on all ofthe samples falling within the linear range of the standard curve. Inthe case of the LANCE assay, themanufacturers report a high-affinity in-teraction between cAMP and the antibody, with IC50s in the lownanomolar range (PerkinElmer Life & Analytical Sciences, 2014). Thestandard curve generates a sigmoidal response curve with a robustspan, but with a relatively limited dynamic range. For example, in the
43M.R. Hunter, M. Glass / Journal of Pharmacological and Toxicological Methods 71 (2015) 42–45
data presented for standard curvemeasurement on a Victor (Fig. 2, page17, (PerkinElmer life & Analytical Sciences, 2014)), the assay has a spanof approximately 16,000 counts and an IC50 of 2.4 nM, but the linearrange of the standard curve (between 10 and 90%) occurs betweenapproximately 0.3 nM and 25 nM. From our own laboratory's experi-ence with this assay, we have found that despite substantial optimisa-tion of conditions including cell number and duration of incubation,data frequently fell on the non-linear portion of the curve, thereby ren-dering the results meaningless. As the method is an “add and read”approach (which exposes the total cAMP in the well to the antibody)there is no scope within this assay design to reprobe samples whichfall outside the linear range. Furthermore, the assay requires cAMP tobe generated in an HBSS buffer, on cells in suspension and at room tem-perature. These are markedly different conditions to that used in mostreceptor signalling assays, therefore reducing the comparability of re-sults generated across a range of assays. The ability to compare resultsacross multiple assays is an increasingly desirable quality, given thestrong interest in functional selectivity between agonist driven path-ways for drug discovery (Gesty-Palmer & Luttrell, 2011).
We have therefore modified the LANCE protocol to enable it to beutilised on cell lysates. This approach has several advantages. Firstly,as only a small proportion of the lysate generated per well is assayed,samples falling outside of the range of the standard curve can be re-assayed at an appropriate dilution to ensure that they fall within thelinear range. Furthermore, the assay can be carried out on adherentcells, and is not sensitive to the conditions under which the cells arestimulated, providing the flexibility to match conditions (stimulationmedia, cell confluency, time and temperature) to other in-house signal-ling assays. The assay as described also allows cell lysates to be frozenprior to detection, allowing samples from multiple days to be detectedsimultaneously without loss of cAMP detection.
2. Materials and methods
2.1. Reagents
Cells were grown and assayed in culture reagents (DMEM, FBS, BSA)supplied by Life Technologies (USA) or Sigma (Australia), on Corningplasticware (Corning, USA). Drugs (isobutylmethylxanthine (IMBX),forskolin, CP55,940) were obtained from Tocris Bioscience (UK) andSigma Aldrich (Australia). The LANCE cAMP kit and half-area white96-well plates were purchased from Perkin Elmer (USA). Additional“detection buffer”was made as described in LANCE cAMP kit handbook(50mMHEPES, 10mMCaCl2, 0.35%TritonX-100, distilledwater and pHadjusted to 7.4).
2.2. Cell stimulation and lysate preparation
Themethodwas developed utilisingHEK 293 cells stably transfectedwith the human CB1 receptor, N-terminally tagged with 3-hemagglutinin as previously described (HEK-hCB1) (Cawston et al.,
Table 1Optimisation considerations for assay conditions.
Condition Optimisation considerations
Assay vessel Well/plate size, volume of growth mediaCell adherence conditions Surface coating of assay vessel, time to adhereConfluency Balance between cAMP concentration in lysate, and effect
of cell contact inhibition on receptor functionMedia Serum starve length, assay buffer/media composition
Drug stimulation conditions Presence and concentration of IBMX, presence andconcentration of forskolin, length of drug incubation,temperature.
Lysis buffer Volume of lysis buffer
2013). Cells were grown in standard growth media consisting ofDMEM supplemented with 10% FBS, as well as 250 ug/ml zeocin as aselection antibiotic, at 37 °C in 5% CO2. Assay conditions were basedon those previously utilised for assaying CB1 receptors by radioimmu-noassay (Kearn, Blake-Palmer, Daniel, Mackie, & Glass, 2005).
Precise plating and stimulation conditions should be optimised foreach cell type. A summary of optimisation considerations is detailed inTable 1. In this case, HEK-hCB1 cells were seeded in poly-L-lysine-treated 96-well plates to achieve 70–80% confluence after 24 h (approx-imately 60,000 cells/well).
Growthmediawas removed fromHEK-hCB1 cells and replacedwithDMEM containing 5 mg/ml BSA and 0.5 mM IBMX, and the cells weresubsequently incubated at 37 °C in 5% CO2 for 30 min immediately be-fore drug stimulation to allow time for a stable base line to develop inthe absence of full serum. BSA was included as a carrier molecule dueto the lipophilic nature of cannabinoid ligands, and could be omittedfor more hydrophyllic ligands.
Drugs were diluted in DMEM containing 5 mg/ml BSA and 0.5 mMIBMX. As CB1 is a Gi-linked receptor, cAMP synthesis was stimulatedwith forskolin to enable the inhibition of this response to be detected.Drugs were diluted to 2× the desired final concentration and added di-rectly to the cells to minimise cell disruption. Cells were stimulated for20 min at 37 °C, and plates were placed on ice immediately thereafterto prevent further signalling. The stimulation medium was quickly re-moved from all wells and the cells were immediately lysed with ice-cold detection buffer (provided in the kit). This step should be optimisedfor each cell type/receptor assayed to ensure that the level of cAMP iswithin the dynamic range of the standard curve,with a suggested startingpoint of 25–50 μl per well in a 96-well format.
Once thedetection buffer is applied, the cells should be gently agitat-ed at 4 °C for 15–30 min to ensure full cell lysis.
2.3. Detection of cAMP
The standard curve dilutions were prepared as described in theLANCE protocol. Briefly, at least six standards with final concentrationsof between 1 μM and 10 pM of cAMPwere prepared in detection bufferfrom the 50 μM cAMP standard supplied in the LANCE kit. In order toutilise the entire antibody component of the kit using the lysate tech-nique, additional detection buffer is required. This can be made in-house, as described in the “Reagents” section above.
The proportion of sample to antibody was based on the suggestedpreparation of the standard curve. Thus, 6 μl of lysate sample or cAMPstandard was transferred to a half-volume white 96-well plate (PerkinElmer). Anti-cAMP antibody, diluted 1/100 in detection buffer (as perkit instructions),was then added immediately to eachwell (6 μl of dilut-ed anti-cAMP antibody/well). The lysate samples and antibody weremixed in the plate and incubated, protected from light, for 30 minprior to the addition of the detection mix (gentle rocking or agitationis optional). The detection mix (europium-streptavidin and biotin-cAMP) was prepared as per LANCE kit instructions (first, intermediate
General notes
Dependent on cell lineHigher cell density gives higher cAMP concentrations in a fixed volume ofdetection buffer.Dependent on cell line/receptor requirements. Generally a serum-free period of30 min to 1 h results in low base line cAMP.These conditions should be determined by the purpose of the experiment. Allother detection conditions can be optimised around these factors.
Lower volume of lysis buffer gives a higher concentration of cAMP in lysate. Lysisbuffer should cover the entire surface of the assay plate to ensure complete lysis.
15000
20000
25000
30000
35000
40000
-11 -10 -9 -8 -7 -60
log [cAMP], MBas
al, 30
ul
FSK, 30u
l
Basal,
50ul
FSK, 50u
l
cAMP standard curve cell lysate samplesA
B Basal (fmol of cAMP per well)
Forskolin stimulated (fmol of cAMP per well)
Apparent fold stimulation
30µl lysate 9.453±3.516 230.22±40.98 24.3550µl lysate 2.7975±0.7825 209.2±23.155 74.78
cou
nts
Fig. 1. Representative data showing the effect of altering lysis buffer volume on cAMP levelsin cell lysates. Cells were plated at the same confluency, stimulated and then lysed. A) Thegraph shows the standard curve, as well as raw data counts for cells stimulated with 1 μMforskolin and then lysed in either 30 μl or 50 μl of lysis buffer. B) Comparison of the apparenteffect of forskolin, using the cAMP concentrations as interpolated from the standard curve.cAMP values are presented as fmol of cAMP per well, as calculated by adjusting for thelysis volume and dilution in the detection mix. All data is shown as mean ± SEM.
log [CP55,940], M
0
50
100
150
-11 -10 -9 -8 -7 -6Basal FSK
fmo
l cA
MP
in w
ell
Fig. 2. Inhibition of forskolin-stimulated cAMP accumulation in HEK-HA-hCB1 cells,utilising themodified LANCE assay. Representative data from one of three separate exper-iments, with each drug condition assayed in triplicate and plotted as mean ± SEM.
44 M.R. Hunter, M. Glass / Journal of Pharmacological and Toxicological Methods 71 (2015) 42–45
dilutions of europium-streptavidin at 1/18, and biotin-cAMP at 1/6;then each intermediate dilution is further diluted 1/125 in detectionbuffer to give the detection mix), and complexes were allowed toform for at least 15 min at room temperature. When the detectionmix had incubated for at least 15 min, and the lysate-antibody sampleshad been incubated for 30min, 12 μl of the detectionmixwas added perwell. The plate was then incubated for a further 60 min (or up to over-night) at room temperature (with optional gentle agitation), before de-tection in a TR-FRET-capable plate reader as per the manufacturer'sinstructions. In these experiments, we have utilised a Wallac Victor1420 Multilabel Counter (Perkin Elmer), with excitation at 340 nmand emission at 615 nm and 665 nm.
2.4. Stability of lysate during freeze–thaw
Onepotential advantage of a lysate-based detectionmethod is that itenables the samples to be reprobed if the initially-detected value fallsoutside of the standard curve. For this to be practical, lysates need tobe stable following freeze–thaw. To test this, cells were stimulated asdescribed above with forskolin, and some lysate immediately assayedfor cAMP. The remaining lysate was frozen at −80 °C for 7 days,warmed to room temperature (with gentle agitation) and re-assayed.
3. Results
3.1. Optimisation of cell lysis volume
HEK-hCB1 cells were incubated in the presence and absence of 1 μMforskolin. Following removal of media, the cells were lysed in either 30or 50 μl of detection buffer and assayed as described above. As this is acompetition assay, increasing cAMP levels result in a decreased TR-FRET signal. The cAMP standard curve data was graphed as a sigmoidalcurve, using GraphPad Prism 6 software, and unknowns were automat-ically interpolated from the standard curve. As shown in Fig. 1A, a typi-cal standard curve spanned approximately 17,000 fluorescent units,with an IC50 of 1.2 nM. The linear portion of the curve (between 10and 90%) therefore ranged from 0.13 nM to 10.8 nM of cAMP per sam-ple. The cell lysate samples were interpolated from the standard curveand adjusted to account for the lysate volume; absolute cAMP per wellis shown in Fig. 1B. As can be seen in Fig. 1A, with a lysate volume of50 μl, the basal concentration of cAMP falls outside of the linear rangeof the curve; however Prism did return interpolated values for 2 of the3 replicates (Prism will not interpolate if the value falls above the“top” or below the “bottom” of the curve, although this does not reflectthe actual dynamic range ofmost assays). As can be seen from the table,while the stimulated levels of cAMP are both within the range of thestandard curve and comparable when estimated by either method, thebasal value for 50 μl lysates is considerably underestimated by thismethod, demonstrating the importance of ensuring that samples fallwithin the linear range. As lysis in 30 μl of detection buffer producedlevels of cAMPwithin the linear range of the curve, thiswas used for fur-ther experiments.
3.2. Determination of receptor-mediated cAMP signalling
To ensure that this detection method maintained sufficient sensitiv-ity to be useful in typical cAMP signalling assays, a concentration re-sponse curve for a cannabinoid receptor agonist was carried out. Cellswere treated with 1 μM forskolin and varying concentrations of theCB1 agonist CP55,940, and the cell lysates were then assayed using thelysate-modified LANCE cAMP assay. As demonstrated in Fig. 2, aconcentration-dependent inhibition of forskolin-stimulated cAMPaccu-mulation was detected with pEC50 for CP55,940 of 9.48 ± 0.09(mean ± standard error of the mean, n = 3), similar to the results re-cently reported for these same cells utilising a cAMP biosensor (pEC509.4 ± 0.1, (Cawston et al., 2013)).
3.3. Determination of lysate stability through freeze–thaw
In order to determine the stability of cAMP in the lysis buffer, cellswere treated with forskolin (1 μM) as above, and the resulting lysatewas probed twice: first immediately after cell lysis and again after7 days storage at −80 °C. Paired T-test analysis showed no statisticaldifference between cAMP concentrationmeasured before or after freez-ing (p N 0.05, n = 3).
4. Discussion
Here we have presented a simple modification of the LANCE cAMPdetection kit, which adds increased flexibility and versatility to thekit's standard applications. This method modification process has also
45M.R. Hunter, M. Glass / Journal of Pharmacological and Toxicological Methods 71 (2015) 42–45
been applied in-house to the AlphaScreen cAMP assay kit (PerkinElmer) (unpublished data), and can presumably be applied to othercommercially-available “add and read” detection systems in order to in-crease reliability and/or cost-effectiveness.
All of the available competition assays require conversion to absolutecAMP levels through the use of a standard curve for accurate data analysis(see Hill et al., 2010 for a discussion on the potential for datamisinterpre-tation from direct detection of cAMP without reference to a standardcurve). As such, these assays are highly reliant on data falling within thedynamic range of the standard curve. Here, we have shown that a rela-tively low concentration of forskolin (1 μM) utilises over 50% of the avail-able dynamic range (Fig. 1), and under our standardised assay conditionsall forskolin concentrations above 3 μM resulted in raw data which wasoutside the dynamic range of the assay (data not shown). However, it iscommon practise to use concentrations of forskolin well above 5 μM forHEK 293 cells, for example (Bitterman, Ramos-Espiritu, Diaz, Levin, &Buck, 2013; Cawston et al., 2013; Wang, Yan, Zheng, He, & Yang, 2014;Wehbi et al., 2013). Therefore, assay design should consider the expectedquantity of cAMP generated. Studies activating Gs-linked GPCRs may re-quire the use of different dilution factors for basal and stimulated condi-tions to gain accurate measures of their actual cAMP levels—somethingwhich could not be achieved in the standard protocol of an “add andread”. Our method modification therefore overcomes what is probablythe greatest limitation of the LANCE cAMP kit (and other similar kits), inthat it allows samples to be diluted and reprobed if they fall outside ofthe standard curve, without the need to repeat the experiment. Impor-tantly, we have shown here that samples can be stored frozen withoutloss of detectable signal. Thiswould also enable samples prepared on sep-arate days to be analysed together for convenience and efficiency, a mea-surewhichwould also conserve reagents because a single standard curvecould be utilised for multiple experiments. The ability to reprobe samplesalso means that this method requires considerably less optimisation thanis required for the standard application of this kit, which requiresmultipleexperiments to establish the conditions that produce signals within thedynamic range. Additionally, the small volumes utilised in the detection(24 μl total) are suitable for detection in 384 well plates, allowing forhigher throughput or automation.
In this example, we have utilised HEK293 cells transfected with thecannabinoidCB1 receptor, but the lysate-modified LANCE cAMPdetectionmethod is adaptable for any adherent cell type, with minimal optimisa-tion from existing protocols set up in any laboratory. This technique alsopermits the use of any cell culture medium during cell stimulation, be-cause media is removed and washed off; therefore the detection methodis not affected bymedia pigments such as phenol red. Indeed, completing
cell stimulation before the detection steps allows researchers to test awide range of experimental paradigms (e.g. salt concentrations) withoutconcern as to whether these will compromise the subsequent detectionassay. Increasingly, pharmacological studies have focused on the “func-tional selectivity” of ligands—this being the relative effect of ligands ondifferent signalling pathways mediated by the same receptor (Kenakin,2011). Such comparisons are substantially more valid if cell stimulationprotocols are kept as similar as possible across different assays, whichthis adaptation makes feasible, as the choice of stimulation buffer andtemperature will not alter the ability to detect signal.
In summary, we show here a straightforward adaptation of themanufacturer's recommendation for a commonly used cell signallingassay, which would improve the utility of the cAMP detection kit.
Acknowledgements
This research was supported by a Royal Society of New ZealandMarsden (#11-UOA-201) Fund Grant to MG.
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