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Calcium flux in MC4-expressing Chem-3 cell line induced by human recombinant NDP-MSH. MC4-expressing Chem-3 cells were loaded w...
ChemiScreen™ Human Recombinant MC4 Melanocortin Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiScreen™ Human Recombinant MC4 Melanocortin Receptor Calcium-Optimized Stable Cell Line
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Full-length human MC4R cDNA encoding for MC4 gene
Background Information:
The melanocortins, α-, β- and -melanocyte-stimulating hormones (MSHs), are peptides derived from a precursor protein POMC. The MSH peptides bind to a family of five Gs-coupled seven transmembrane recptors (MC1-5) and play important roles in energy balance, reproductive function, pigmentation and inflammation (Gantz and Fong, 2003). The activity of the melanocortins is modulated by endogenous antagonist proteins, agouti and agouti-related protein (AGRP). MC4 is expressed primarily in the CNS and appears to play a prominent role in energy homeostasis. The hypothalamus, which is a major central site for control of feeding behavior, prominently expresses MC4. Targeted deletion of MC4 in mice and naturally occurring mutations in MC4 in humans result in obese phenotypes. In addition, blockade of MC4 function by antagonists and targeted deletion of the gene in mice reverses melanocortin agonist-induced inhibition of food intake and promotes weight gain in uremia and cancer-induced cachexia (Cheung et al., 2005; Markison et al., 2005). Chemicon's cloned human MC4-expressing cell line is made in the Chem-3 host, which supports high levels of recombinant MC4 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 MC4 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.
UniProt Summary:
FUNCTION: SwissProt: P32245 # Receptor specific to the heptapeptide core common to adrenocorticotropic hormone and alpha-, beta-, and gamma-MSH. This receptor is mediated by G proteins that stimulate adenylate cyclase.
SIZE: 332 amino acids; 36943 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Brain, placental, and gut tissues.
DISEASE:SwissProt: P32245 # Defects in MC4R are a cause of autosomal dominant obesity [MIM:601665].
SIMILARITY: SwissProt: P32245 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 332 amino acids; 36943 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Brain, placental, and gut tissues.
DISEASE:SwissProt: P32245 # Defects in MC4R are a cause of autosomal dominant obesity [MIM:601665].
SIMILARITY: SwissProt: P32245 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Brand Family:
Chemicon
Protein Target:
MC4
Presentation:
Cells are frozen at 2 x 106 cells/mL in RPMI/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.
Ligand Type:
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 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 conical tube containing 10 mL Chem-3 Growth Media. Centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 20 mL Chem-3 Growth Media. Transfer to a T75 flask and place the flask in a humidified incubator at 37°C with 5% CO2.
3. Maintain the cells in suspension between 0.1 x 106 and 1 x 106 cells/mL. Cells are typically passaged 1:10 every 2-3 days, or 1:20 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator. Chem-3 cells tend to proliferate rapidly, so care should be taken not to allow them to become overconfluent.
4. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count cells. Centrifuge cells at 200 x g for 5 min. Resuspend cells at 2-5 x 106 cells/mL in Chem-3 Freezing Media. 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.
5. Cells should be passaged at least once after thawing prior to use in calcium flux assays.
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 conical tube containing 10 mL Chem-3 Growth Media. Centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 20 mL Chem-3 Growth Media. Transfer to a T75 flask and place the flask in a humidified incubator at 37°C with 5% CO2.
3. Maintain the cells in suspension between 0.1 x 106 and 1 x 106 cells/mL. Cells are typically passaged 1:10 every 2-3 days, or 1:20 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator. Chem-3 cells tend to proliferate rapidly, so care should be taken not to allow them to become overconfluent.
4. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count cells. Centrifuge cells at 200 x g for 5 min. Resuspend cells at 2-5 x 106 cells/mL in Chem-3 Freezing Media. 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.
5. Cells should be passaged at least once after thawing prior to use in calcium flux assays.
Target Sub-Family:
Melanocortin
GPCR Class:
A
Promotional Text:
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Gene Symbol:
- MC4R
- MC4-R
- MGC138197
- MGC126851
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiScreen™ Human Recombinant MC4 Melanocortin Receptor Calcium-Optimized Stable Cell Line
Materials Required but Not Delivered:
Chem-3 Growth Media:
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate (Millipore SLM-140)
10% heat-inactivated FBS
100 U/ml each penicillin and streptomycin (from 100x stock, Millipore TMS-AB2-C)
1% Chem-3 Growth Supplement (Millipore catalog # HTSCHEM-3S)
G-418/Geneticin (600ug/mL)
Note: The Chem-3 Growth Supplement is required for growth of Chem-3 cell lines
Chem-3 Plating Media:
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate
10% heat-inactivated FBS
100 U/ml each penicillin and streptomycin
Chem-3 Freezing Media:
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate
20% heat-inactivated FBS
10% DMSO (cell culture grade)
Several calcium-sensing dyes are available that eliminate the need for washing. Any of these dyes may be used by the manufacturer's protocol, with the following recommendations for suspension cells such as Chem-3:
1. Cells should be passaged at least once after thawing, and should be in mid-log phase of growth at the time of the assay (0.8-1 x 106 cells/mL). Propagate desired number of cells; 2 x 105 cells/well are recommended for the assay. If the cell density exceeds 1 x 106 cells/mL or the media is becoming yellow, passage the cells again and culture for at least one day before proceeding.
2. On the day of the assay, count cells and collect the desired number of cells. Centrifuge cells and resuspend at 2 x 106 cells/ml in FLIPR assay buffer. Plate 50µL of cell suspension/well (96-well plate) and allow cells time to settle and equilibrate to new buffer environment at 37°C for at least one hour.
3. Prepare a 2x dye loading solution. After the cell incubation (step 2) is complete, add 50µL/well of the 2x dye loading solution for a final volume of 100µL. Incubate the cells at 37°C in the dark for 30 - 60 minutes.
4. For FLIPR ligand addition, set pipet tip height at 125 µL and dispense rate of 25 µL/sec.
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate (Millipore SLM-140)
10% heat-inactivated FBS
100 U/ml each penicillin and streptomycin (from 100x stock, Millipore TMS-AB2-C)
1% Chem-3 Growth Supplement (Millipore catalog # HTSCHEM-3S)
G-418/Geneticin (600ug/mL)
Note: The Chem-3 Growth Supplement is required for growth of Chem-3 cell lines
Chem-3 Plating Media:
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate
10% heat-inactivated FBS
100 U/ml each penicillin and streptomycin
Chem-3 Freezing Media:
RPMI-1640 containing 0.3 g/L L-glutamine, no pyruvate
20% heat-inactivated FBS
10% DMSO (cell culture grade)
Several calcium-sensing dyes are available that eliminate the need for washing. Any of these dyes may be used by the manufacturer's protocol, with the following recommendations for suspension cells such as Chem-3:
1. Cells should be passaged at least once after thawing, and should be in mid-log phase of growth at the time of the assay (0.8-1 x 106 cells/mL). Propagate desired number of cells; 2 x 105 cells/well are recommended for the assay. If the cell density exceeds 1 x 106 cells/mL or the media is becoming yellow, passage the cells again and culture for at least one day before proceeding.
2. On the day of the assay, count cells and collect the desired number of cells. Centrifuge cells and resuspend at 2 x 106 cells/ml in FLIPR assay buffer. Plate 50µL of cell suspension/well (96-well plate) and allow cells time to settle and equilibrate to new buffer environment at 37°C for at least one hour.
3. Prepare a 2x dye loading solution. After the cell incubation (step 2) is complete, add 50µL/well of the 2x dye loading solution for a final volume of 100µL. Incubate the cells at 37°C in the dark for 30 - 60 minutes.
4. For FLIPR ligand addition, set pipet tip height at 125 µL and dispense rate of 25 µL/sec.
Concentration:
2 x 106 cells/vial
Quality Assurance:
SPECIFICATIONS: EC50 for calcium mobilization by NDP-MSH: ~ 1 μM
Cell Type:
Chem-3
Cell Line Type:
GPCR Cell Lines
Host Cells:
Chem-3
UniProt Number:
Entrez Gene Number: