ChemiSCREEN™ Human Recombinant EP4 Prostandoid Receptor Calcium-Optimized Ready-to-Assay Cells
Description:
ChemiSCREEN™ Human Recombinant EP4 Prostandoid Receptor Calcium-Optimized Ready-to-Assay Cells
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.
Prostanoids are a series of arachidonic acid metabolites produced by the action of cyclooxygenase and further modified by isomerases and synthases. Cells rapidly secrete prostanoids after synthesis, whereupon the prostanoids bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001). The prostaglandin PGE2 causes pain, vasodilation, immunosuppression of T cells, bone remodeling and promotion of carcinogenesis. Four related GPCRs, EP1, EP2, EP3 and EP4, each bind to PGE2, but the different G protein coupling status of each receptor leads to distinct biological effects. EP4 couples primarily to Gs to increase intracellular cAMP levels. During neonatal development, EP4 participates in closure of the ductus arteriosus, a process required for switching circulation from the placenta to the lungs (Nguyen et al., 1997). In addition, EP4 mediates PGE2-induced bone formation by promoting osteoblastogenesis, and selective EP4 agonists are being evaluated as potential treatments for osteoporosis (Yoshida et al., 2002). Millipore's cloned human EP4-expressing cells are made in the Chem-1 host, an adherent cell line. The untreated EP4-Chem-1 cell line and the Ready-To-Assay EP4 cells have equivalent EC50s for PGE2.
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.
Prostanoids are a series of arachidonic acid metabolites produced by the action of cyclooxygenase and further modified by isomerases and synthases. Cells rapidly secrete prostanoids after synthesis, whereupon the prostanoids bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001). The prostaglandin PGE2 causes pain, vasodilation, immunosuppression of T cells, bone remodeling and promotion of carcinogenesis. Four related GPCRs, EP1, EP2, EP3 and EP4, each bind to PGE2, but the different G protein coupling status of each receptor leads to distinct biological effects. EP4 couples primarily to Gs to increase intracellular cAMP levels. During neonatal development, EP4 participates in closure of the ductus arteriosus, a process required for switching circulation from the placenta to the lungs (Nguyen et al., 1997). In addition, EP4 mediates PGE2-induced bone formation by promoting osteoblastogenesis, and selective EP4 agonists are being evaluated as potential treatments for osteoporosis (Yoshida et al., 2002). Millipore's cloned human EP4-expressing cells are made in the Chem-1 host, an adherent cell line. The untreated EP4-Chem-1 cell line and the Ready-To-Assay EP4 cells have equivalent EC50s for PGE2.
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 cells/vial
Gene Symbol:
- PTGER4
- MGC126583
- EP4
- EP4R
- PTGER2
Analytes Available:
EP4
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 PGE2; | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 4.48 nM | 1297 | 0.82 |
| Continuous Passage Cells | 4.44 nM | 2094 | 0.78 |
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 37°C 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 37°C 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.