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Calcium flux in NMU2–expressing Chem-1 cell line induced by NmU-25. NMU2 expressing Chem-1 cells and Wild-Type Chem-1 cells (WT, Millipore catalog # ...
ChemiSCREEN™ Human Recombinant NMU2 Neuromedin U Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant NMU2 Neuromedin U Receptor Calcium-Optimized Stable Cell Line
Trade Name:
Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Plasmid encoding full-length human NMUR2 cDNA encoding NMU2.
The stable clonal cell line was selected by resistance to geneticin/G418 followed by limited dilution cloning. The cell line was tested and found to have equivalent EC50 and signal greater than 1000 at 1, 3 and 6 weeks of continuous culture.
The stable clonal cell line was selected by resistance to geneticin/G418 followed by limited dilution cloning. The cell line was tested and found to have equivalent EC50 and signal greater than 1000 at 1, 3 and 6 weeks of continuous culture.
Background Information:
Neuromedin U (NmU) is a peptide which regulates peripheral functions such as smooth muscle contraction and blood pressure, and CNS functions including nociception and feeding activity (Brighton et al., 2004a). Two GPCRs, NMU1 and NMU2, mediate the contractile effects of neuromedin U by activation of both Gq and Gi (Brighton et al., 2004b). Compared to the wide distribution of NMU1 in peripheral tissue, expression of NMU2 receptor is limited to areas of the brain, such as the paraventricular nucleus, along the wall of the third ventricle in the hypothalamus and the CA1 region of the hippocampus, and to the spinal cord (Howard et al. 2000). Recent study has shown that mice deficient in NMU2 but not NMU1 receptor had impaired nociceptive responses suggesting that the pro-nociceptive effects of NmU in mice appear to be mediated through NMU2 (Zeng et al., 2006; Torres et al. 2007). Millipore’s cloned human NMU2-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant NMU2 expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists at NMU2.
Application Notes:
Calcium flux assay, ligand binding assays
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.
Species:
Human
Quality Assurance:
Table I. Comparison of potency values of NMU2-expressing Chem-1 cells with values described in the literature.
| ligand | assay | potency (nM) | Reference |
|---|---|---|---|
| NmU-25 | Calcium | EC50 = 3.1 | Figure 1 |
| NmU-25 | Calcium | EC50 = 5 | Shan et al., 2000 |
| NmU-25 | Calcium | EC50 = 2.4 | Raddatz et al., 2000 |
| NmU-25 | Calcium | EC50 = 0.43 | Brighton et al., 2004b |
| NmU-25 | Radioligand binding with [125I]-NmU-25 | Kd = 0.112 | Brighton et al., 2004b |
Brand Family:
Chemicon
Host Cells:
Chem-1
Presentation:
Cells are frozen at 2 x 106 cells/mL in 90% fetal bovine serum/10% DMSO. Cell line tests negative for mycoplasma.
Protein Target:
NMU2
Ligand Type:
peptide
Storage Conditions:
1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen.
2. 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. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37ºC with 5% CO2.
3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
4. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca++ and Mg++ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37ºC with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Chem-1 Growth Media per 1 mL trypsin.
5. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
6. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 106 cells/mL in Chem-1 Freezing Media (cell densities of 2-10 x 106 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen.
7. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Chem-1 Plating Media for plating for calcium assay.
2. 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. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37ºC with 5% CO2.
3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
4. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca++ and Mg++ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37ºC with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Chem-1 Growth Media per 1 mL trypsin.
5. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
6. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 106 cells/mL in Chem-1 Freezing Media (cell densities of 2-10 x 106 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen.
7. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Chem-1 Plating Media for plating for calcium assay.
Target Sub-Family:
Neuromedin U
Promotional Text:
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GPCR Class:
A
Packaging:
2 x 106 cells/vial
Product Name:
ChemiSCREEN™ Human Recombinant NMU2 Neuromedin U Receptor Calcium-Optimized Stable Cell Line
Entrez Gene Number:
Materials Required but Not Delivered:
Chem-1 Growth 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)
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250 μg/mL Genetecin/G-418
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
Chem-1 Freezing Media:
90% heat-inactivated FBS
10% DMSO (cell culture grade)
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)
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250 μg/mL Genetecin/G-418
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
Chem-1 Freezing Media:
90% heat-inactivated FBS
10% DMSO (cell culture grade)