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Calcium flux in NK3R-expressing Chem-1 cell line induced by Neurokinin B. NK3-expressing Chem-1 cells and Wild-Type Chem-1 cells (Chemicon...
ChemiSCREEN™ Human Recombinant NK3 Tachykinin Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant NK3 Tachykinin Calcium-Optimized Stable Cell Line
Trade Name:
Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Full-length human TACR3 cDNA encoding NK3
Background Information:
NK3 is one of three mammalian G-protein coupled receptors subtypes (NK1, NK2, NK3) that mediate the biological effects of the tachykinin peptide family. Neurokinin B is the tachykinin family peptide with the highest affinity to NK3R, but it still induces a response with NK1, and NK2 with lower potency (Pennefather et al., 2004; Regoli et al., 1994; Maggi et al., 1993). NK3, like the other tachykinin receptors, signals through Gq to stimulate intracellular calcium (Pinnock et al., 1994). Mammalian tachykinins are present in the brain and gut. NK3 receptor antagonists reduce intestinal motility and nociception in animal models of gastrointestinal disorders, and may be effective in irritable bowel syndrome (Sanger 2004). Chemicon's cloned human NK3-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant NK3 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to enhance coupling the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between NK3 and its ligands.
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.
Entrez Gene Summary:
This gene belongs to a family of genes that function as receptors for tachykinins. Receptor affinities are specified by variations in the 5'-end of the sequence. The receptors belonging to this family are characterized by interactions with G proteins and 7 hydrophobic transmembrane regions. This gene encodes the receptor for the tachykinin neurokinin 3, also referred to as neurokinin B.
UniProt Summary:
FUNCTION: SwissProt: P29371 # This is a receptor for the tachykinin neuropeptide neuromedin-K (neurokinin B). It is associated with G proteins that activate a phosphatidylinositol-calcium second messenger system.
SIZE: 465 amino acids; 52202 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
PTM: The anchoring of this receptor to the plasma membrane is probably mediated by the palmitoylation of a cysteine residue.
SIMILARITY: SwissProt: P29371 ## Belongs to the G-protein coupled receptor 1 family.
MISCELLANEOUS: The rank order of affinity of this receptor to tachykinins is: neuromedin-K > substance K > substance P.
SIZE: 465 amino acids; 52202 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
PTM: The anchoring of this receptor to the plasma membrane is probably mediated by the palmitoylation of a cysteine residue.
SIMILARITY: SwissProt: P29371 ## Belongs to the G-protein coupled receptor 1 family.
MISCELLANEOUS: The rank order of affinity of this receptor to tachykinins is: neuromedin-K > substance K > substance P.
Species:
Human
Quality Assurance:
EC50 for calcium mobilization by Neurokinin B: ~ 0.69 nM
Cell Type:
Chem-1
Cell Line Type:
GPCR Cell Lines
Brand Family:
Chemicon
Host Cells:
Chem-1
Protein Target:
NK3
Format:
Cell Line
UniProt Number:
Ligand Type:
peptide
Target Sub-Family:
Tackykinin /neurokinin
Storage Conditions:
1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen. Maintain frozen in liquid nitrogen for up to 5 years.
2. 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. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37°C 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 37°C 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 37°C 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 37°C 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 37°C 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.
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GPCR Class:
A
Packaging:
2x106 cells/vial
Gene Symbol:
- TACR3
- MGC148061
- TAC3R
- TAC3RL
- MGC148060
- NKR
- NK-3R
- NK3R
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiSCREEN™ Human Recombinant NK3 Tachykinin 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)
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)
250μg/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:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)
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)
250μg/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:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)