A1R expressing cells labeled with Terbium

Tag-lite cells transiently expressing the A1 receptor labeled with Terbium for use in receptor binding applications. The cells are provided ready to use.

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  • Fully functional cell line Fully functional cell line
  • Transient expression Transient expression
  • Cellular binding assay Cellular binding assay

Tag-lite cells transiently expressing the A1 receptor labeled with Terbium for use in receptor binding applications. The cells are provided ready to use.

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Overview

A1 receptor is one of the four adenosine receptor family (including A2A, A2B and A3 receptors).  It has vital role in cardiac and renal processes. It regulates neurotransmitters in the brain, such as dopamine and glutamate. Thus, it is considered as an interesting target in treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.

Cells expressing the A1 receptor are provided pre-labeled with Terbium, and can be used to conduct receptor binding studies on the aforementioned receptor.

Benefits

  • PRE-LABELED WITH TERBIUM
  • FROZEN CELLS, READY TO BE USED
  • CELLS/LIGAND VALIDATED FOR BINDING

Assay principle

Running your A1 receptor binding assay using Tag-lite is as easy as it can get. Simply dispense 10 µL of labeled cells (C1TT1A1) into each well, followed by 5 µL of labeled ligand (L0067RED) and 5 µL of the compound you wish to test. Like all HTRF assays, Tag-lite assays do not require any washing steps. A diagram of the procedure to be followed is given on the right.

Diagram of Adenosine A1 receptor binding assay using the Tag-lite protocol

Saturation binding (KD)

A saturation binding assay measures total and non-specific binding for increasing concentrations of ligand under equilibrium conditions. To perform the assay, the fluorescent ligand is titrated into a solution containing a fixed amount of labeled cells, and then incubated to equilibrium. The HTRF Ratio obtained from this titration is the total binding.

Diagram of a saturation binding assay using Tag-lite
Representative data obtained when running a saturation binding assay

Watch this videoexplaining how to run a saturation binding assay using Tag-lite.

Competitive binding (KI)

A competitive binding assay is performed to measure the dissociation constant, Ki. To perform the assay, the compound is titrated into a solution containing a fixed concentration of fluorescent ligand and a fixed amount of cells.
Diagram of a competitive binding assay using Tag-lite
Representative data obtained when running a competitive binding assay
Watch this video explaining how to run a competitive binding assay using Tag-lite.

Kd and Ki validation

Examples of data obtained using Adenosine A1 receptor labeled cells and their matching fluorescent ligand (L0067RED). DPCPX (8-Cyclopentyl-1,3-dipropylxanthine) was used for Ki determination in the competitive binding assay. Results may vary from one HTRF® compatible reader to another.

kd determination using A1 labeled cells and their matching fluorescent ligand L0067RED
ki determination using A1 labeled cells and L0067RED, DPCPX was used as ref compound

Compound profiling on Adenosine A1, A2A, A2B and A3 receptors

Adenosine receptors designated by four subtypes A1, A2A, A2B and A3 were shown to be promising pharmacological targets for the development of therapeutic compounds for various therapeutic areas such as cancer, neurodegenerative disease and COPD/Asthma. Thus, a wide range of compounds, and particularly antagonists have been largely studied.

The Tag-Lite® binding assay platform offers the complete adenosine receptors subset A1, A2A, A2B and A3 to characterize compounds in term of binding affinity (Ki) and selectivity profile.

The set of compounds have been profiled on A1, A2A, A2B and A3 receptors using their respective fluorescent ligands L0067RED, L0058RED, L0068RED and L0069GRE.The selectivity profiles of several compounds involved in different therapeutic areas were assessed with this Tag-Lite platform.


The table below summarizes the resulting Ki obtained for each receptor subtype for the full panel of compounds. The results are confirming the expected selectivity and the range of affinities for these reference compounds.

CompoundPharmacological classCompounds affinities (Ki - nM) for the different adenosine receptor subtypes


A1A2AA2BA3
XACNon selective antagonist12152050
Theophylline Non selective antagonist4000500018000140000
DPCPXA1 antagonist7.6170145800
VipadenantA2A antagonist700.95520000
Ciforadenant (CPI-444)A2A antagonist1503.46601600
Preladenant (SCH 420814)A2A antagonist> 20 0005> 20 000>20 000
Istradefylline (KW-6002)A2A antagonist>10 00040>10 000>10 000
ZM241385A2A antagonist2600.740750
AZD4635A2A antagonist90015660> 10 000
MRS1706A2B inverse agonist15032530> 300
MRS1220A3 antagonist32537> 5001.6
Adenosine A1 - Assay validation: DPCPX (A1 selective compound) profiling on Adenosine receptors
Adenosine A1 - Assay validation: Preladenant (A2A selective compound) profiling on Adenosine receptors
Adenosine A1 - Assay validation: MRS1706 (A3 selective compound) profiling on Adenosine receptors

The graphs show the pattern of the dose-response curves obtained for five compounds displaying different selectivity profile.

Adenosine A1 - Assay validation: MRS1220 (A3 selective compound) profiling on Adenosine receptors
Adenosine A1 - Assay validation: Theophylline (non selective compound) profiling on Adenosine receptors
Cisbio Product Catalog 2019

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