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Screen Quest™ HEK-CNGC-Chemokine (C-X-C motif) Receptor 4 Cells

Screen Quest™ Live Cell cAMP Assay Principle
Screen Quest™ Live Cell cAMP Assay Principle
Screen Quest™ Live Cell cAMP Assay Principle
<strong>Figure 1</strong>. <strong>Response of HEK-CXCR4 cells and parental cells to SDF-1&alpha;.</strong><br>Both CXCR4 cells and parental cells were plated overnight in 20 &micro;L/well culture medium in a Poly-D-Lysine coated black wall/clear bottom 384-well plate. The cells were incubated with equal volume (20 &micro;L) of MP dye working solution at room temperature for 2 hours. Two readings were obtained prior to and 25 min after the addition of SDF-1&alpha;. Ratios of the two readings (F/F0) are plotted in the figure.<br>A. Dose response curve of SDF-1&alpha; in HEK-CXCR4 cell line. EC50 = 14 ng/ml in the presence of PDE inhibitor Ro20-1724 and &beta;-adrenoceptor agonist isoproterenol<br>B: Parental cells do not respond to SDF-1&alpha;.
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageLiquid nitrogen
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


G protein coupled receptors (GPCRs) are one of the largest receptor classes targeted by drug discovery programs. Calcium flux (coupled via Gq pathway) assay is a preferred method in drug discovery for screening GPCR targets. However, over 60% of the known GPCRs signal through adenylyl cyclase activity coupled to cAMP. Most of the existing cAMP assays not only require cell lysis but also lack both temporal and spatial resolution. Screen Quest™ cell lines enable to investigate GPCRs that do not conventionally couple through intracellular calcium. Screen Quest™ cell lines are based on a series of G-protein chimeras, including the promiscuous G-protein, Gα16, and an exogenous cyclic nucleotide-gated channel (CNGC). The chimeras consist of the alpha subunit of a Gq-protein complex whose 5 carboxy-terminal amino acids have been replaced with those from one of the other G-proteins (either Gαs, Gαi, Gαo, or Gαz). These amino acids are responsible for the coupling of the receptor to its G-protein. Co-expression of these chimeras or CNGC with specific non-Gq-coupled receptors may result in the generation of an intracellular calcium signal upon receptor stimulation. Screen Quest™ HEK-CNGC-Chemokine (C-X-C motif) Receptor 4 (CXCR4) cell line is HEK-293 cells stably transfected with both the CNGC and human Chemokine (C-X-C motif) Receptor 4. The constitutively expressed CNGC in the cells responds in real-time to increases or decreases in intracellular cAMP levels by coordinately altering cation flux (e.g., calcium, potassium or sodium). Activation of the CXCR4 in these cells by specific ligands can be detected with either a calcium-sensitive fluorescent indicator (such as Calbryte 520 AM, Cal-520 AM, Fluo-8 AM, or Fluo-4 AM and the corresponding no wash calcium kit) or an AAT’s optimized membrane-potential assay kit. This cell line has been successfully used in drug discovery and screening environments for studying GPCRs that do not conventionally couple through intracellular calcium. It has been effectively used with FLIPR and FDSS systems.

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Citations


View all 2 citations: Citation Explorer
Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling
Authors: Avanzato, D and Genova, T and Pla, A Fiorio and Bernardini, M and Bianco, S and Bussolati, B and Mancardi, D and Giraudo, E and Maione, F and Cassoni, P and others, undefined
Journal: Scientific Reports (2016)
The M2 muscarinic receptors are essential for signaling in the heart left ventricle during restraint stress in mice
Authors: Tomankova, Hana and Valuskova, Paulina and Varejkova, Eva and Rotkova, Jana and Benes, Jan and Myslivecek, Jaromir
Journal: Stress (2015)

References


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