ChemiSCREEN™ Human Recombinant CRF1 Neuropeptide Receptor Calcium-Optimized Ready-to-Assay Cells
Description:
ChemiSCREEN™ Human Recombinant CRF1 Neuropeptide Receptor Calcium-Optimized Ready-to-Assay Cells
Trade Name:
ChemiScreen
Product Overview:
Full-length human CRF1 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 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.
The CRF1 receptor is a Gs-coupled GPCR expressed in the brain and pituitary gland that binds to several neuropeptides, including corticotropin-releasing factor (CRF) and urocortin, and the amphibian peptide sauvagine (Chen et al., 1993; Dautzenberg and Hauger, 2002; Bale and Vale, 2004). CRF plays a predominant role in stress response mediated by the hypothalamic-pituitary-adrenal axis, and alterations in CRF and its receptors CRF1 and CRF2 appear to be linked to depression and anxiety (Holsboer, 1999; Bale and Vale, 2004). A number of small molecule antagonists of the CRF1 receptor have been characterized, including R121919, SC241, NBI27914, antalarmin, DMP-696, and CP 154,526. When delivered in animal models of psychiatric disorders, these antagonists display effectiveness in reducing stress-related behaviors (Kehne and De Lombaert, 2002). Millipore's cloned human CRF1-expressing cells are made in the Chem-1 host, an adherent cell line. The untreated CRF1-Chem-1 cell line and the Ready-To-Assay CRF1 cells have equivalent EC50s for Sauvagine.
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.
The CRF1 receptor is a Gs-coupled GPCR expressed in the brain and pituitary gland that binds to several neuropeptides, including corticotropin-releasing factor (CRF) and urocortin, and the amphibian peptide sauvagine (Chen et al., 1993; Dautzenberg and Hauger, 2002; Bale and Vale, 2004). CRF plays a predominant role in stress response mediated by the hypothalamic-pituitary-adrenal axis, and alterations in CRF and its receptors CRF1 and CRF2 appear to be linked to depression and anxiety (Holsboer, 1999; Bale and Vale, 2004). A number of small molecule antagonists of the CRF1 receptor have been characterized, including R121919, SC241, NBI27914, antalarmin, DMP-696, and CP 154,526. When delivered in animal models of psychiatric disorders, these antagonists display effectiveness in reducing stress-related behaviors (Kehne and De Lombaert, 2002). Millipore's cloned human CRF1-expressing cells are made in the Chem-1 host, an adherent cell line. The untreated CRF1-Chem-1 cell line and the Ready-To-Assay CRF1 cells have equivalent EC50s for Sauvagine.
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.
Cell Line Type:
Ready-to-Assay
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:
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020)
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 penicillin and streptomycin (from 100x stock, Millipore TMS-AB2-C)
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020)
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 penicillin and streptomycin (from 100x stock, Millipore TMS-AB2-C)
Cell Type:
Chem-1
Gene Symbol:
- CRHR1
- CRF1
- CRFR
- CRHR1f
- CRHR
- CRFR1
- CRF-R
- CRH-R1h
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 Sauvagine; | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 4.2nM | 3175 | 0.7 |
| Continuous Passage Cells | 11.5 nM | 4788 | 0.6 |
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 Chem-1 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 Chem-1 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 Chem-1 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 Chem-1 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.