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Figure 1. Calcium flux in IP1-expressing Chem-1 cell line induced by Iloprost. IP1 expressing Chem-1 cells and Wild-Type Chem-...
ChemiSCREEN™ Human Recombinant IP1 Prostanoid Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant IP1 Prostanoid Receptor Calcium-Optimized Stable Cell Line
Trade Name:
Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Full-length human PTGIR cDNA encoding IP1
Background Information:
Prostacyclin (PGI2) is released by vascular endothelial cells and serves as a potent vasodilator, inhibitor of platelet aggregation, and moderator of vascular smooth muscle cell proliferation-migration-differentiation (Narumiya et al. 1999). The function of protacyclin is mediated via a seven transmembrane GPCR, IP1, which is known to couple to Gs and Gq signaling pathways. Mice lacking the IP1 receptor have shown increased susceptibility to thrombosis (Murata et al. 1997), enhanced injury-induced vascular proliferation and platelet activation (Cheng et al. 2002), as well as reperfusion injury (Xiao et al. 2001). The recent world-wide withdrawal of selective COX-2 inhibitors, rofecoxib (Vioxx™) and valdecoxib (Bextra™), is also due to their discriminating suppression of COX-2-derived prostacyclin and IP1-mediated cardioprotective effects, leading to increased risk of cardiovascular events (Fitzgerald 2004). Millipore's cloned human IP1-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant IP1 expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between IP1 and its ligands.
Application Notes:
Calcium flux assay, 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:
The protein encoded by this gene is a member of the G-protein coupled receptor family 1 and has been shown to be a receptor for prostacyclin. Prostacyclin, the major product of cyclooxygenase in macrovascular endothelium, elicits a potent vasodilation and inhibition of platelet aggregation through binding to this receptor.
UniProt Summary:
FUNCTION: SwissProt: P43119 # Receptor for prostacyclin (prostaglandin I2 or PGI2). The activity of this receptor is mediated by G(s) proteins which activate adenylate cyclase.
SIZE: 386 amino acids; 40956 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
PTM: Palmitoylation of either Cys-308 or Cys-311 is sufficient to maintain functional coupling to G(s) and signaling. & Isoprenylation does not influence ligand binding but is required for efficient coupling to the effectors adenylyl cyclase and phospholipase C.
SIMILARITY: SwissProt: P43119 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 386 amino acids; 40956 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
PTM: Palmitoylation of either Cys-308 or Cys-311 is sufficient to maintain functional coupling to G(s) and signaling. & Isoprenylation does not influence ligand binding but is required for efficient coupling to the effectors adenylyl cyclase and phospholipase C.
SIMILARITY: SwissProt: P43119 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Quality Assurance:
EC50 for calcium mobilization by Iloprost: ~53nM
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:
IP1 / PGI2
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:
Prostanoid
Promotional Text:
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GPCR Class:
A
Packaging:
2 x 106 cells/vial
Gene Symbol:
- PTGIR
- PRIPR
- MGC102830
- IP
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiSCREEN™ Human Recombinant IP1 Prostanoid 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-A)
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)
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250µg/mL Genetecin/G-418
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
Chem-1 Freezing Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020-A)
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)
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250µg/mL Genetecin/G-418
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
Chem-1 Freezing Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)