READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS HUMAN RECOMBINANT CXCR2 RECEPTOR
Description:
READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS HUMAN RECOMBINANT CXCR2 RECEPTOR
Product Overview:
Full-length human CXCR2 cDNA
Background Information:
Millipore’s Ready-To-AssayTM 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-AssayTM cells are derived from ChemiScreenTM 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-AssayTM 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-AssayTM cells is identical to that of the originating GPCR cell line.
CXCR2 is a 7-TM G-protein coupled receptor that binds to the chemokines GROα, GROβ, GROγ, IL-8, ENA-78, NAP-2 and GCP-2 (Olson and Ley, 2002). Neutrophils, mast cells and microvascular endothelial cells express CXCR2, and interactions of CXCR2 with its ligands promotes chemotaxis of these cell types (Heidemann et al., 2003; Nilsson et al., 1999; White et al., 1998). Studies with mice lacking CXCR2 indicate that CXCR2 promotes growth of primary tumors and secondary metastases (Keane et al., 2004), and plays an essential role in hyperoxia-induced lung injury (Sue et al., 2004). In addition, cytomegalovirus encodes a CXCR2-binding chemokine, vCXC-1, that promotes neutrophil migration to infected cells (Penfold et al., 1999). Millipore’s cloned CXCR2-expressing cell line is made in the Chem-1 host, an adherent cell line that supports high levels of recombinant CXCR2 expression on the cell surface and contains high levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. The untreated CXCR2-Chem-1 cell line and the Ready-To-AssayTM CXCR2 cells have equivalent EC50s for IL-8.
The Ready-To-AssayTM cells are derived from ChemiScreenTM 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-AssayTM 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-AssayTM cells is identical to that of the originating GPCR cell line.
CXCR2 is a 7-TM G-protein coupled receptor that binds to the chemokines GROα, GROβ, GROγ, IL-8, ENA-78, NAP-2 and GCP-2 (Olson and Ley, 2002). Neutrophils, mast cells and microvascular endothelial cells express CXCR2, and interactions of CXCR2 with its ligands promotes chemotaxis of these cell types (Heidemann et al., 2003; Nilsson et al., 1999; White et al., 1998). Studies with mice lacking CXCR2 indicate that CXCR2 promotes growth of primary tumors and secondary metastases (Keane et al., 2004), and plays an essential role in hyperoxia-induced lung injury (Sue et al., 2004). In addition, cytomegalovirus encodes a CXCR2-binding chemokine, vCXC-1, that promotes neutrophil migration to infected cells (Penfold et al., 1999). Millipore’s cloned CXCR2-expressing cell line is made in the Chem-1 host, an adherent cell line that supports high levels of recombinant CXCR2 expression on the cell surface and contains high levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. The untreated CXCR2-Chem-1 cell line and the Ready-To-AssayTM CXCR2 cells have equivalent EC50s for IL-8.
Application Notes:
Calcium flux assay
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 a promiscuous G-protein.
Packaging:
1 x 107 cells/vial
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:
- IL8RB
- CD182
- CMKAR2
- CXCR-2
- IL8RA
- IL8R2
- CXCR2
- CDw128b
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 IL-8 (nM) | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 2.22 | 3468 | 0.55 |
| Continuous Passage Cells | 0.73 | 5013 | 0.80 |
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.