READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT EP2 PROSTANOID RECEPTOR
Description:
READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT EP2 PROSTANOID RECEPTOR
HUMAN RECOMBINANT EP2 PROSTANOID RECEPTOR
Trade Name:
Chemicon (Millipore)
Product Overview:
Full-length human PTGER2 cDNA encoding EP2
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 bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001). The protanoid PGE2 causes pain, vasodilation, immunosuppression of T cells, bone resorption 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. EP2 couples primarily to Gs to increase intracellular cAMP levels. Mice deficient in EP2 receptor showed impaired ovulation and fertilization, salt-sensitive hypertension (Kennedy et al., 1999). It has been shown that EP2 receptors are also involved in cancer associated immunodeficiency. Thus, genetic knockout of the EP2 receptor reduced tumor growth and prolonged survival in mice that had undergone isograft injection of MC26 or Lewis lung carcinoma cells (Yang et al., 2003). Millipore’s cloned EP2-expressing cell line is made in the Chem-9 host, an adherent cell line that supports high levels of recombinant EP2 expression on the cell surface and contains high levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. The untreated EP2-Chem-9 cell line and the Ready-To-Assay™ EP2 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 bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001). The protanoid PGE2 causes pain, vasodilation, immunosuppression of T cells, bone resorption 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. EP2 couples primarily to Gs to increase intracellular cAMP levels. Mice deficient in EP2 receptor showed impaired ovulation and fertilization, salt-sensitive hypertension (Kennedy et al., 1999). It has been shown that EP2 receptors are also involved in cancer associated immunodeficiency. Thus, genetic knockout of the EP2 receptor reduced tumor growth and prolonged survival in mice that had undergone isograft injection of MC26 or Lewis lung carcinoma cells (Yang et al., 2003). Millipore’s cloned EP2-expressing cell line is made in the Chem-9 host, an adherent cell line that supports high levels of recombinant EP2 expression on the cell surface and contains high levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. The untreated EP2-Chem-9 cell line and the Ready-To-Assay™ EP2 cells have equivalent EC50s for PGE2.
Application Notes:
Calcium flux assay
Species Reactivity:
Human
Host Cells:
Chem-9, an adherent cell line expressing the promiscuous G-protein.
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:
- PTGER2
- EP2
Analytes Available:
EP2
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 (nM) | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 30.7 | 1532 | 0.63 |
| Continuous Passage Cells | 25.5 | 2120 | 0.74 |
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.