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Figure 1. Calcium flux in GPR99-expressing Chem-1 cell line induced by α-ketoglutarate. GPR99–expressing Chem-1 cells were loaded with Fluo-4 and ca...
ChemiSCREEN™ Human Recombinant GPR99 α Ketoglutarate Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant GPR99 α Ketoglutarate Receptor Calcium-Optimized Stable Cell Line
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Full-length human OXGR1 cDNA encoding GPR99
Background Information:
GPR99 (also known as GPR80) was originally identified as an orphan with a sequence most closely related to P2Y receptors. Studies to deorphanize GPR99 revealed that α-ketoglutarate activates GPR99 through Gq to increase intracellular calcium (He et al., 2004; Qi et al., 2004; Abbracchio et al., 2005). The EC50 of GPR99 for α -ketoglutarate is in the µM range, similar to the concentration of α-ketoglutarate in the circulation, indicating that the interaction is physiologically significant. Chemicon's cloned human GPR99-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant GPR99 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to enhance coupling of the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between GPR99 and its ligands.
Application Notes:
Calcium flux assays, ligand binding assays
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:
G protein-coupled receptors (GPCRs, or GPRs) contain 7 transmembrane domains and transduce extracellular signals through heterotrimeric G proteins.[supplied by OMIM]
UniProt Summary:
FUNCTION: SwissProt: Q96P68 # Receptor for alpha-ketoglutarate. Seems to act exclusively through a G(q)-mediated pathway (By similarity).
SIZE: 337 amino acids; 38251 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Detected in kidney and, to a lower extend, in placenta. Not detected in brain tissues including the frontal cortex, caudate putamen, thalamus, hypothalamus, hippocampus or pons.
SIMILARITY: SwissProt: Q96P68 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 337 amino acids; 38251 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Detected in kidney and, to a lower extend, in placenta. Not detected in brain tissues including the frontal cortex, caudate putamen, thalamus, hypothalamus, hippocampus or pons.
SIMILARITY: SwissProt: Q96P68 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Quality Assurance:
EC50 for calcium mobilization by α-ketoglutarate: ~ 260 µM
Cell Type:
Chem-1
Cell Line Type:
GPCR Cell Lines
Brand Family:
Chemicon
Host Cells:
Chem-1
Presentation:
Cells are frozen at 2 x 106 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.
Protein Target:
OXGR1 , GPR99 , GPR80
UniProt Number:
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 37°C water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37°C with 5% CO2.
3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
4. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca++ and Mg++ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37°C with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Chem-1 Growth Media per 1 mL trypsin.
5. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
6. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 106 cells/mL in Chem-1 Freezing Media (cell densities of 2-10 x 106 are also acceptable if necessary). 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.
7. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Chem-1 Plating Media for plating for calcium assay.
2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37°C water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37°C with 5% CO2.
3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
4. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca++ and Mg++ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37°C with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Chem-1 Growth Media per 1 mL trypsin.
5. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
6. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 106 cells/mL in Chem-1 Freezing Media (cell densities of 2-10 x 106 are also acceptable if necessary). 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.
7. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Chem-1 Plating Media for plating for calcium assay.
Target Sub-Family:
alpha-Ketoglutarate
Promotional Text:
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GPCR Class:
A
Packaging:
2 x 106 cells/vial
Gene Symbol:
- OXGR1
- GPR80
- P2RY15
- P2Y15
- GPR99
- MGC119206
- MGC119208
- MGC119207
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiSCREEN™ Human Recombinant GPR99 α Ketoglutarate Receptor Calcium-Optimized Stable Cell Line
Entrez Gene Number:
Materials Required but Not Delivered:
Chem-1 Growth Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020)
10% heat-inactivated FBS
1x Nonessential amino acids (from 100x stock, Millipore TMS-001-C)
10mM HEPES (from 1 M HEPES, Millipore TMS-003-C)
100 U/mL penicillin and streptomycin (from 100x stock, Millipore TMS-AB2-C)
250 µg/mL G-418/Geneticin
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
100 U/mL penicillin and streptomycin
Chem-1 Freezing Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
100 U/mL penicillin and streptomycin
10% DMSO (cell culture grade)
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020)
10% heat-inactivated FBS
1x Nonessential amino acids (from 100x stock, Millipore TMS-001-C)
10mM HEPES (from 1 M HEPES, Millipore TMS-003-C)
100 U/mL penicillin and streptomycin (from 100x stock, Millipore TMS-AB2-C)
250 µg/mL G-418/Geneticin
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
100 U/mL penicillin and streptomycin
Chem-1 Freezing Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
100 U/mL penicillin and streptomycin
10% DMSO (cell culture grade)