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Figure 1. Calcium flux in A3-expressing Chem-3 cell line induced Cl-IB-MECA. A3-expressing Chem-3 cells and wild-type (WT) Che...
Figure 2. Assay for antagonist activity at A3 by calcium flux assay. A3-expressing Chem-3 cells were loaded with Fluo-4 and wa...
ChemiSCREEN™ Human Recombinant A3 Adenosine Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant A3 Adenosine Receptor Calcium-Optimized Stable Cell Line
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Full-length human ADORA3 cDNA encoding A3
Background Information:
Extracellular adenosine mediates a multitude of biological effects, including wakefulness, antiarrythmia, bronchoconstriction and response to ischemia and oxidative stress. A family of four GPCR adenosine receptors, A1, A2A, A2B and A3, is responsible for these effects (Fredholm et al., 2001). A3, which couples to Gi/o, is expressed in mast cells along with A2B. Mice lacking A3 display reduced mast cell degranulation and bronchoconstriction in response to adenosine (Tilley et al., 2003; Zhong et al., 2003). Chemicon's cloned human A3-expressing cell line is made in the Chem-3 host, which supports high levels of recombinant A3 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists at A3.
Application Notes:
Calcium flux assay, ligand binding assays
Species Reactivity:
Human
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Entrez Gene Summary:
This gene encodes a protein that belongs to the family of adenosine receptors, which are G-protein-coupled receptors that are involved in a variety of intracellular signaling pathways and physiological functions. The receptor encoded by this gene mediates a sustained cardioprotective function during cardiac ischemia, it is involved in the inhibition of neutrophil degranulation in neutrophil-mediated tissue injury, it has been implicated in both neuroprotective and neurodegenerative effects, and it may also mediate both cell proliferation and cell death. Multiple transcript variants encoding different isoforms have been found for this gene.
UniProt Summary:
FUNCTION: SwissProt: P33765 # Receptor for adenosine. The activity of this receptor is mediated by G proteins which inhibits adenylyl cyclase. Possible role in reproduction.
SIZE: 318 amino acids; 36185 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
PTM: Phosphorylation on Ser-317 may be crucial for rapid desensitization (By similarity).
SIMILARITY: SwissProt: P33765 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 318 amino acids; 36185 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
PTM: Phosphorylation on Ser-317 may be crucial for rapid desensitization (By similarity).
SIMILARITY: SwissProt: P33765 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Brand Family:
Chemicon
Protein Target:
A3
Presentation:
Cells are frozen at 2 x 106 cells/mL in RPMI/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.
Ligand Type:
non-peptide
Storage Conditions:
1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen. Maintain frozen in liquid nitrogen for up to 5 years.
2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37ºC water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a conical tube containing 10 mL Chem-3 Growth Media. Centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 20 mL Chem-3 Growth Media. Transfer to a T75 flask and place the flask in a humidified incubator at 37ºC with 5% CO2.
3. Maintain the cells in suspension between 0.1 x 106 and 1 x 106 cells/mL. Cells are typically passaged 1:10 every 2-3 days, or 1:20 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator. Chem-3 cells tend to proliferate rapidly, so care should be taken not to allow them to become overconfluent.
4. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count cells. Centrifuge cells at 200 x g for 5 min. Resuspend cells at 2-5 x 106 cells/mL in Chem-3 Freezing Media. Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen.
5. Cells should be passaged at least once after thawing prior to use in calcium flux assays.
2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37ºC water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a conical tube containing 10 mL Chem-3 Growth Media. Centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 20 mL Chem-3 Growth Media. Transfer to a T75 flask and place the flask in a humidified incubator at 37ºC with 5% CO2.
3. Maintain the cells in suspension between 0.1 x 106 and 1 x 106 cells/mL. Cells are typically passaged 1:10 every 2-3 days, or 1:20 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator. Chem-3 cells tend to proliferate rapidly, so care should be taken not to allow them to become overconfluent.
4. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count cells. Centrifuge cells at 200 x g for 5 min. Resuspend cells at 2-5 x 106 cells/mL in Chem-3 Freezing Media. Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen.
5. Cells should be passaged at least once after thawing prior to use in calcium flux assays.
Target Sub-Family:
Adenosine
GPCR Class:
A
Promotional Text:
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Gene Symbol:
- ADORA3
- AD026
- bA552M11.5
- ORFNames=UNQ1931/PRO4406
- RP11-552M11.7
- A3AR
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiSCREEN™ Human Recombinant A3 Adenosine Receptor Calcium-Optimized Stable Cell Line
Materials Required but Not Delivered:
Chem-3 Growth Media:
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate (Millipore SLM-140)
10% heat-inactivated FBS
100 U/ml each penicillin and streptomycin (from 100x stock, Millipore TMS-AB2-C)
1% Chem-3 Growth Supplement (Millipore catalog # HTSCHEM-3S)
G-418/Geneticin (600ug/mL)
Note: The Chem-3 Growth Supplement is required for growth of Chem-3 cell lines
Chem-3 Plating Media:
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate
10% heat-inactivated FBS
100 U/ml each penicillin and streptomycin
Chem-3 Freezing Media:
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate
20% heat-inactivated FBS
10% DMSO (cell culture grade)
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate (Millipore SLM-140)
10% heat-inactivated FBS
100 U/ml each penicillin and streptomycin (from 100x stock, Millipore TMS-AB2-C)
1% Chem-3 Growth Supplement (Millipore catalog # HTSCHEM-3S)
G-418/Geneticin (600ug/mL)
Note: The Chem-3 Growth Supplement is required for growth of Chem-3 cell lines
Chem-3 Plating Media:
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate
10% heat-inactivated FBS
100 U/ml each penicillin and streptomycin
Chem-3 Freezing Media:
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate
20% heat-inactivated FBS
10% DMSO (cell culture grade)
Quality Assurance:
EC50 for calcium mobilization by Cl-IB-MECA: 14 nM
IC50 for VUF5574: 15 nM
IC50 for VUF5574: 15 nM
Cell Type:
Chem-3
Cell Line Type:
GPCR Cell Lines
Host Cells:
Chem-3
Format:
Cell Line
UniProt Number:
Packaging:
2 x 106 cells/vial
Entrez Gene Number: