LANCE cAMP assays

 

Overview

We currently offer two LANCE kits for cAMP detection: the LANCE Ultra cAMP kit, and the LANCE cAMP kit. This page contains information about the LANCE cAMP kit. You can also view information on the LANCE Ultra cAMP kit.

The LANCE® cAMP assay is a homogeneous time-resolved fluorescence energy transfer (TR-FRET) immunoassay designed to measure cAMP produced upon modulation of adenylyl cyclase activity by GPCRs. The assay is based on the competition between a Europium-labeled cAMP tracer complex and sample cAMP for binding sites on cAMP-specific antibodies labeled with Alexa Fluor 647 dye. The Europium-labeled cAMP tracer complex is formed by the tight interaction between biotin-cAMP and Eu-streptavidin.

When antibodies are bound to the Eu-streptavidin/biotin-cAMP tracer, excitation at 340 nm excites the Europium. The energy is transferred to the Alexa Fluor-labeled antibody. The fluorescence measured at 665 nm will decrease in the presence of cAMP from test samples, and resulting signals will be inversely proportional to the cAMP concentration of a sample (this is a competition assay) (Figure 1). The assay is intended for the detection of cAMP produced by cells or cell membrane preparations stimulated with GPCR agonists.

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Figure 1. LANCE cAMP competition assay principle.

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Gs-coupled receptors

For Gs-coupled receptors, agonist stimulation of cells or membrane preparations results in an increase in cAMP levels, and a decrease in assay signal (competition assay). Addition of an antagonist will reverse the response, resulting in an increase in signal.

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Gi-coupled receptors

For Gi-coupled receptors, cells are simultaneously stimulated by the adenylyl cyclase activator, forskolin, and by the agonist. An agonist will inhibit the forskolin-induced cAMP production, resulting in an increase in signal compared to forskolin alone (competition assay). Antagonists will block the effect of the agonist, resulting in a decrease in signal toward forskolin-induced levels.

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What do I need to run this assay?

Required reagents available from 爱游戏平台注册登录 :

  • LANCE cAMP kit (catalog number AD0262, AD0262E, AD0263, or AD0264)
  • Microplates (we recommend our standard white OptiPlates™ or white ProxiPlates™ - one of each of these plates, 384-well, is included in the AD0262E kit)
  • TopSeal™-A adhesive plate seal
  • Cell lines (or you can use your own; view 爱游戏平台注册登录 cell lines and membranes)
  • BSA solution (catalog number CR84-100; a small vial is included in the AD0262E kit)


Required reagents available from various suppliers (see where 爱游戏平台注册登录 R&D gets these reagents):

  • Versene™ for cell detachment
  • Appropriate media
  • Other components to make stimulation buffer (1X HBSS, 5 mM HEPES)
  • IBMX
  • Forskolin (if studying Gi-coupled receptors)


Instrumentation/Equipment:

  • A TRF-capable plate reader
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Protocol-in-brief

The general protocol for the LANCE cAMP assay is summarized in Figure 2. Please refer to the manual for more-detailed information.

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Figure 2. General protocol for the LANCE cAMP assay.

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Assay optimizations

  1. IBMX concentration
  2. Cell number
  3. Forskolin concentration (for Gi-coupled receptors)
  4. Agonist concentration
  5. Stimulation time
  6. Read time
  • Refer to pages 20-22 of the LANCE cAMP Manual for more information.

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Application notes, posters, and guides

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Citations

View a brief list of LANCE cAMP citations.

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Tips and FAQs

  • Note that you cannot use 10X LANCE Detection buffer (#CR97-100) in a LANCE cAMP assay. The Detection buffer included in the LANCE cAMP kits is a different formulation, that contains detergent for cell lysis.

Q. Do I have to lyse my cells?
A. The Detection buffer contains a detergent (Triton X-100) that will lyse most cells.


Q. Do I need to use IBMX?
A. If you are using cells, yes. IBMX is a phosphodiesterase inhibitor and prevents the degradation of cAMP.


Q. Why am I using forskolin?
A. If you are studying a Gi-coupled receptor, you will be looking for a decrease in cAMP levels upon stimulation of your receptor. To raise the baseline levels of cAMP for unstimulated cells, you will treat all samples with forskolin. Forskolin stimulates adenylyl cyclase, which will trigger cAMP production.


Q. How do I pick a forskolin concentration?
A. When you run your forskolin titration curve, you will have a graph that shows forskolin concentration vs. counts (similar to Figure 3). What you want to do is pick a concentration of forskolin that matches the EC50 or EC80 from your cAMP standard curve. To do this, you will have to convert your forskolin concentrations to the amount of cellular cAMP. You can do this by standardizing the signals from your forskolin titration and the signals from your cAMP standard titration. Then find the forskolin concentration where your signal matches the EC50 or EC80 concentration from your cAMP standard curve.

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Figure 3. Adherent cell LANCE cAMP assay in B2-AR/CHO cells, 24 µL total volume. Twenty four hours prior to the LANCE experiment, the cells grown in a T-75 flask were detached. Cells were counted and plated in a CulturPlate™-384 from 爱游戏平台注册登录 (catalog number 6007680) at 8,000 cells/well with 50 µL of culture media. Since the CulturPlate-384 plate is opaque, 8,000 cells were also plated in clear-bottom Falcon 384-well plates in parallel to assess cell density and monitor growth. Approximately 70% confluency was attained on the experiment day. Protocol was as indicated in the manual.


Q. I think the biology will be more relevant if I treat my adherent cells when they are attached, rather than resuspended. Can I do this?
A. Yes. You can run the entire assay in a TC-treated plate. Plate your cells in a TC-treated microplate, add test compounds in stimulation buffer, and then follow the protocol as normal. See Figure 3 above.

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