READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT IP1 PROSTANOID RECEPTOR
Description:
READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT IP1 PROSTANOID RECEPTOR
HUMAN RECOMBINANT IP1 PROSTANOID RECEPTOR
Product Overview:
Full-length human human PTGIR cDNA encoding IP1
Background Information:
Millipore’s Ready-To-Assay™ Calcium-Optimized Cells are GPCR-expressing cell lines that are designed for simple, rapid calcium assays with no requirement for culturing cells. The user simply thaws the cells with maximal viability, dispenses into assay plates, and assays for calcium response the next day.
The Ready-To-Assay™cells are derived from ChemiScreen™ calcium-optimized stable cell lines, which express the GPCR target of interest at high levels on the cell surface, in a host cell line containing high levels of the promiscuous Gα15 protein to couple the receptor to the calcium signaling pathway. The Ready-To-Assay™ cells are prepared by chemical treatment at a concentration optimized for effective growth arrest while maintaining high viability (>80%) after thawing and overnight plating. Pharmacological functionality of the Ready-To-Assay™ cells is identical to that of the originating GPCR cell line.
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 cloned human IP1-expressing cell line is made in the Chem-1 host, an adherent cell line that supports high levels of recombinant IP1 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. The untreated IP1-Chem-1 cell line and the Ready-To-Assay™ IP1 cells have equivalent EC50s for Iloprost.
The Ready-To-Assay™cells are derived from ChemiScreen™ calcium-optimized stable cell lines, which express the GPCR target of interest at high levels on the cell surface, in a host cell line containing high levels of the promiscuous Gα15 protein to couple the receptor to the calcium signaling pathway. The Ready-To-Assay™ cells are prepared by chemical treatment at a concentration optimized for effective growth arrest while maintaining high viability (>80%) after thawing and overnight plating. Pharmacological functionality of the Ready-To-Assay™ cells is identical to that of the originating GPCR cell line.
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 cloned human IP1-expressing cell line is made in the Chem-1 host, an adherent cell line that supports high levels of recombinant IP1 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. The untreated IP1-Chem-1 cell line and the Ready-To-Assay™ IP1 cells have equivalent EC50s for Iloprost.
Application Notes:
Calcium flux assay
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.
Host Cells:
Chem-1, an adherent cell line expressing the promiscuous G-protein, Gα15
Packaging:
1 x 107 viable cells/mL
Materials Required but Not Delivered:
PLATING 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)
100 U/mL Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
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)
100 U/mL Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
Gene Symbol:
- PTGIR
- PRIPR
- MGC102830
- IP
Analytes Available:
IP1
Presentation:
Cells are frozen at 1 x 107 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO.
Quality Assurance:
| EC50 for Iloprost (nM) | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 12 | 5167 | 0.54 |
| Continuous Passage Cells | 22 | 6154 | 0.47 |
Detection Methods:
Radioactive
Kit or Assay Type:
Cell Based Assays
Protein or Enzyme Type:
GPCRs
Species:
Human
Storage Conditions:
Place cells in liquid nitrogen immediately upon receipt. Maintain frozen in liquid nitrogen for up to 5 years.
1) 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.
2) Transfer contents of the vial to a sterile 15 mL conical tube. Add 10 mL prewarmed plating media to the cells and mix gently to resuspend cells. Centrifuge at 200 x g. Remove all but 0.5 mL media.
3) Resuspend cells to 0.5 x 106 cells/mL in plating media. Dispense the cell suspension into a 96-well assay plate at 200 µL per well to obtain a density of approximately 1 x 105 cells/well.
4) Place the assay plate in a humidified 37°C incubator with 5% CO2.
5) The cells may be assayed 16-24 hours after plating. It is recommended to wash the cells with assay buffer at least once prior to addition of loading dye.
1) 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.
2) Transfer contents of the vial to a sterile 15 mL conical tube. Add 10 mL prewarmed plating media to the cells and mix gently to resuspend cells. Centrifuge at 200 x g. Remove all but 0.5 mL media.
3) Resuspend cells to 0.5 x 106 cells/mL in plating media. Dispense the cell suspension into a 96-well assay plate at 200 µL per well to obtain a density of approximately 1 x 105 cells/well.
4) Place the assay plate in a humidified 37°C incubator with 5% CO2.
5) The cells may be assayed 16-24 hours after plating. It is recommended to wash the cells with assay buffer at least once prior to addition of loading dye.