商品编号


38101



品牌


AAT Bioquest



公司


AAT Bioquest



公司分类


cAMP GPCR Assays



Size

Each


商品信息



Overview



Printer Friendly Version







Ex/Em (nm)
None/None



MW
N/A



CAS #
N/A



Solvent
N/A



Storage

N2 (L)



Category
GPCR
cAMP GPCR Assays




Related




Screen Quest? cell lines are a series of cells that have been successfully used in drug discovery and screening environments for studying G-protein-coupled receptors (GPCR) that do not conventionally couple through intracellular calcium. It have been effectively used with the FLIPR, FDSS Systems in conjunction with non-Gq coupled members of many receptors such as chemokine,
SERO
tonin, glutamate, dopamine, opoid, vasopressin and ?- and ?-adrenoceptor receptor families. Over 60% of the known G-protein-coupled receptors signal through pathways other than Gq which lead to an increase in intracellular calcium, and as genomics reveals more G-protein-coupled receptor targets this trend continues to increase. Screen Quest? cell lines are used for investigating G-protein-coupled receptors (GPCR) 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, G?16. 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 G?s, G?i, G?o, or G?z). These amino acids are respons
IBL
e for the coupling of the receptor to its G-protein. Co-expression of these chimeras with specific non-Gq-coupled receptors which normally act through the cAMP pathway may result in the generation of an intracellular calcium signal upon receptor stimulation. Screen Quest? CHO-Gqi cell line is CHO-K1 cells stably transfected with the chimeric Gqi alpha subunit protein. When used as a host cell for transfection expression of Gi-coupled receptors, the constitutively expressed Gqi protein in the cells allows the transfected receptor which normally act through the cAMP pathway, to couple to Gq signal transduction and mobilized intracellular calcium. Activation of the specific non-Gq-coupled receptors in these cells by specific ligands can be detected using calcium sensitive dyes such as Cal-520 AM, Fluo-8 AM, or Fluo-4 AM.

This protocol only provides a guideline, and should be modified according to your specific needs.
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Cell Density and Storage
The cells are frozen at a density of 2 X 106 cells in 90% fetal bovine serum and 10% (v/v) DMSO. To insure the highest level of vi
ABI
lity, thaw the vial and initiate the culture as soon as poss
IBL
e upon receipt. Cells must be stored in liquid nitrogen if not immediately processed upon receipt.

Cell Culture Medium
Basal medium: Ham’s F12 culture medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ mL penicillin, 100 ?g/mL streptomycin (Basal medium).
Selection Medium: Basal Medium with 200 ?g/mL hygromycin B and 400 ug/mL G418.

Thawing and Seeding Cells

Prepare Basal Medium. Prepare 37 °C Water Bath. Thaw cells rapidly by removing from liquid nitrogen, and immediately immersing in a 37 °C water bath. Remove the vial from the water bath as soon as the contents are thawed, and sterilize the exterior of the vial with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.

Add vial contents to 25 mL cell culture medium in T75 Tissue Culture Treated Flask. Gently swirl flask and place in a humidified, tissue culture incubator, 37 °C, 5% CO2.

All live cells should be attached after 18-24 hours post–thaw. Change Basal Medium with Selection Medium.



Subculture and Propagation

When cells are approximately 80% confluent, passage the cells.

Passage the cells 1:10 every 3-4 days, using Trypsin-EDTA to dissociate the cells.

It is highly recommended that a frozen cell bank be established at low passage number.



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References & Citations




Citation Explorer





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