ChemiSCREEN™ Human Recombinant A2B Adenosine Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant A2B Adenosine Receptor Calcium-Optimized Stable Cell Line
Trade Name:
ChemiScreen
Product Overview:
Full-length human ADORA2B cDNA encoding A2B
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 G protein-coupled adenosine receptors, A1, A2A, A2B and A3, is responsible for these effects. A2B, which couples to Gs, is expressed in smooth muscle of the bowel, and blood vessels, and mediates relaxation of these tissues in response to adenosine (Fredholm et al., 2001). Airway mast cells also express A2B, which appears to contribute to bronchoconstriction during asthma (Holgate, 2005). Chemicon’s cloned human A2B -expressing cell line is made in the Chem-3 host, which supports high levels of recombinant A2B 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 A2B.
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.
Cell Line Type:
GPCR Cell Lines
Host Cells:
Chem-3, a suspension cell line endogenously expressing the promiscuous G protein, Gα15.
Packaging:
2 x 106 cells/vial
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)
Cell Type:
Chem-3
Gene Symbol:
- ADORA2B
- ADORA2
Format:
Cell Line
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.
Quality Assurance:
EC50 for calcium mobilization by NECA: ~ 110 nM
IC50 for MRS 1754 with 2x EC50 NECA: ~141 nM
IC50 for MRS 1754 with 2x EC50 NECA: ~141 nM
Protein or Enzyme Type:
GPCRs
Species:
Human
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.