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Calcium flux in 5-HT2C-expressing Chem-1 cell line induced by Ro 60-0175 and α-methyl-5-hydroxy- tryptamine. 5-HT2C-expressing ...
ChemiScreen™ Human Recombinant 5-HT2C Serotonin Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiScreen™ Human Recombinant 5-HT2C Serotonin Receptor Calcium-Optimized Stable Cell Line
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Full-length unedited human HTR2C cDNA encoding 5-HT2C.
Background Information:
5-Hydroxytryptamine (5-HT, also commonly known as serotonin) is synthesized in enterochromaffin cells in the intestine and in serotonergic nerve terminals. In the periphery, 5-HT mediates gastrointestinal motility, platelet aggregation, and contraction of blood vessels. Many functions of the central nervous system are influenced by 5-HT, including sleep, motor activity, sensory perception, arousal and appetite. A family of 12 GPCRs and one ion channel mediate the biological effects of 5-HT (Hoyer et al., 1994). 5-HT2C, which couples to Gq in most cells to stimulate intracellular calcium, is prominently expressed in brain and appears to modulate depression, anxiety and appetite (Miller, 2005; Serretti et al., 2004; Wood, 2003). The mRNA encoding 5-HT2C undergoes selective RNA editing that changes 4 amino acids in the second intracellular loop; these changes result in alteration of efficiency of coupling to G proteins. Alterations in editing of 5-HT2C have been detected in victims of suicidal depression and in mice treated with the SSRI, fluoxetine (Tohda et al., 2006). Chemicon's cloned human 5-HT2C -expressing cell line is made in the Chem-1 host, which supports high levels of recombinant 5-HT2C expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between 5-HT2C and its ligands.
Application Notes:
Calcium flux assay, ligand binding assays
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.
Entrez Gene Summary:
Serotonin (5-hydroxytryptamine, 5-HT), a neurotransmitter, elicits a wide array of physiological effects by binding to several receptor subtypes, including the 5-HT2 family of seven-transmembrane-spanning, G-protein-coupled receptors, which activate phospholipase C and D signaling pathways. This gene encodes the 2C subtype of serotonin receptor and its mRNA is subject to multiple RNA editing events, where genomically encoded adenosine residues are converted to inosines. RNA editing is predicted to alter amino acids within the second intracellular loop of the 5-HT2C receptor and generate receptor isoforms that differ in their ability to interact with G proteins and the activation of phospholipase C and D signaling cascades, thus modulating serotonergic neurotransmission in the central nervous system. Studies in humans have reported abnormalities in patterns of 5-HT2C editing in depressed suicide victims.
UniProt Summary:
FUNCTION: SwissProt: P28335 # This is one of the several different receptors for 5- hydroxytryptamine (serotonin), a biogenic hormone that functions as a neurotransmitter, a hormone, and a mitogen. This receptor mediates its action by association with G proteins that activate a phosphatidylinositol-calcium second messenger system.
SIZE: 458 amino acids; 51821 Da
SUBUNIT: Interacts with MPDZ.
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
DOMAIN: SwissProt: P28335 The PDZ domain-binding motif is involved in the interaction with MPDZ.
PTM: N-glycosylated.
SIMILARITY: Belongs to the G-protein coupled receptor 1 family.
SIZE: 458 amino acids; 51821 Da
SUBUNIT: Interacts with MPDZ.
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
DOMAIN: SwissProt: P28335 The PDZ domain-binding motif is involved in the interaction with MPDZ.
PTM: N-glycosylated.
SIMILARITY: Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Brand Family:
Chemicon
Protein Target:
5-HT2C
Presentation:
Cells are frozen at 2 x 106 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.
Ligand Type:
non-peptide
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 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 5-HT 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 5-HT 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 5-HT 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 5-HT Chem-1 Plating Media for plating for calcium assay.
Target Sub-Family:
Serotonin
GPCR Class:
A
Promotional Text:
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Gene Symbol:
- HTR2C
- 5-HT-2C
- 5-HT2C
- HTR1C
- 5HT-1C
- 5-HTR2C
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiScreen™ Human Recombinant 5-HT2C Serotonin Receptor Calcium-Optimized Stable Cell Line
Materials Required but Not Delivered:
Chem-1 Growth Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020 or equivalent)
20% heat-inactivated dialyzed FBS (Hyclone SH30079.03)
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)
G-418 (250ug/mL)
Note: Dialyzed FBS is the recommended serum, as undialyzed FBS contains serotonin that can desensitize 5-HT receptors. Cells proliferate more slowly in media containing dialyzed FBS than in media containing undialyzed FBS. If faster cell growth is desired, the 5-HT2C-Chem-1 cells may be propagated in media with undialyzed FBS, but should be returned to media containing dialyzed FBS for at least one passage prior to assay.
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated dialyzed 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 (undialyzed FBS is recommended)
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 or equivalent)
20% heat-inactivated dialyzed FBS (Hyclone SH30079.03)
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)
G-418 (250ug/mL)
Note: Dialyzed FBS is the recommended serum, as undialyzed FBS contains serotonin that can desensitize 5-HT receptors. Cells proliferate more slowly in media containing dialyzed FBS than in media containing undialyzed FBS. If faster cell growth is desired, the 5-HT2C-Chem-1 cells may be propagated in media with undialyzed FBS, but should be returned to media containing dialyzed FBS for at least one passage prior to assay.
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated dialyzed 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 (undialyzed FBS is recommended)
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)
Quality Assurance:
EC50 for calcium mobilization by Ro 60-0175: ~ 510 nM
EC50 for calcium mobilization by α-methyl-5-hydroxytryptamine: ~ 45 nM
EC50 for calcium mobilization by α-methyl-5-hydroxytryptamine: ~ 45 nM
Cell Type:
Chem-1
Cell Line Type:
GPCR Cell Lines
Host Cells:
Chem-1
UniProt Number:
Packaging:
2 x 106 cells/vial
Entrez Gene Number: