READY-TO-ASSAY CALCIUM-OPTIMIZED CELLS HUMAN RECOMBINANT CCR1 CHEMOKINE RECEPTOR
Description:
READY-TO-ASSAY CALCIUM-OPTIMIZED CELLS HUMAN RECOMBINANT CCR1 CHEMOKINE RECEPTOR
Trade Name:
Chemicon (Millipore)
Product Overview:
Full-length human CCR1 cDNA
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 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-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.
CCR1 is a GPCR that binds to a variety of CC ligands, including MIP-1α, RANTES, MCP-3, HCC-1, HCC-2, HCC-4, and MPIF-1 (Olson and Ley, 2002). Lymphocytes, macrophages, dendritic cells, and GM-CSF-activated neutrophils express CCR1 (Kaufmann et al., 2001; Cheng et al., 2001). Two selective, non-peptide small molecule antagonists of CCR1, BX-471 and CP-481,715, have been synthesized (Gladue et al., 2003; Liang et al., 2000). Pharmacological and genetic targeting of CCR1, either alone or in combination with cyclosporin A, reduces cardiac and renal allograft rejection (Gao et al., 2000; Horuk et al., 2001a; Horuk et al., 2001b), allergic encephalomyelitis (Liang et al., 2000), and renal fibrosis (Anders et al., 2002) in experimental models. Millipore's cloned human CCR1-expressing cells are made in the Chem-1 host, an adherent cell line. The untreated CCR1-Chem-1 cell line and the Ready-To-Assay CCR1 cells have equivalent EC50s for MIP-1α.
The Ready-To-Assay 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-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.
CCR1 is a GPCR that binds to a variety of CC ligands, including MIP-1α, RANTES, MCP-3, HCC-1, HCC-2, HCC-4, and MPIF-1 (Olson and Ley, 2002). Lymphocytes, macrophages, dendritic cells, and GM-CSF-activated neutrophils express CCR1 (Kaufmann et al., 2001; Cheng et al., 2001). Two selective, non-peptide small molecule antagonists of CCR1, BX-471 and CP-481,715, have been synthesized (Gladue et al., 2003; Liang et al., 2000). Pharmacological and genetic targeting of CCR1, either alone or in combination with cyclosporin A, reduces cardiac and renal allograft rejection (Gao et al., 2000; Horuk et al., 2001a; Horuk et al., 2001b), allergic encephalomyelitis (Liang et al., 2000), and renal fibrosis (Anders et al., 2002) in experimental models. Millipore's cloned human CCR1-expressing cells are made in the Chem-1 host, an adherent cell line. The untreated CCR1-Chem-1 cell line and the Ready-To-Assay CCR1 cells have equivalent EC50s for MIP-1α.
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.
Descriptive Text:
Host Cells:
Chem-1, an adherent cell line expressing the promiscuous G-protein, Gα15.
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:
- CCR1
- HM145
- CCR-1
- MIP1aR
- RANTES-R
- CKR-1
- CD191
- CMKBR1
- CC-CKR-1
- MIP-1alpha-R
- SCYAR1
- CMKR1
Analytes Available:
CCR1
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 SDF-α | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 4.9 nM | 1940 | 0.69 |
| Continuous Passage Cells | 7.8 nM | 1784 | 0.52 |
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.