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Figure 1. Calcium flux in GLP-1-expressing Chem-9 cell line induced by GLP-1 (7-36) and Exendin 4. GLP-1-expressing Chem-9 cells and wild-type Chem-...
ChemiSCREEN™ Human Recombinant GLP-1 Glucagon-Like Peptide Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant GLP-1 Glucagon-Like Peptide Receptor Calcium-Optimized Stable Cell Line
Trade Name:
Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Full-length human GLP1R cDNA encoding GLP-1
Background Information:
Glucagon-like peptide-I (GLP-1), part of the secretin peptide family, is a class B (class 2) receptor and is involved in glucose-dependent insulin secretion. Insulin secretion is predominantly controlled by glucose levels in the blood, however glucagon stimulates the secretion of insulin (Drucker et al. 1987). The GLP-1 receptor is coupled to Gs resulting in increased cAMP levels (Mayo et al. 2003). GLP-1 has been shown to have an affect in delayed gastric emptying and appetite regulation. Due to the involvement in insulin secretion GLP-1 has been identified as a therapeutic target in type II diabetes (Mayo et al. 2003, D'Alessio et al. 2004). Millipore's cloned human GLP-1 -expressing cell line is made in the Chem-9 host, which supports high levels of recombinant GLP-1 expression on the cell surface and contains high levels of promiscuous G proteins to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between GLP-1 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.
UniProt Summary:
FUNCTION: SwissProt: P43220 # This is a receptor for glucagon-like peptide 1. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase.
SIZE: 463 amino acids; 53060 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: P43220 ## Belongs to the G-protein coupled receptor 2 family.
SIZE: 463 amino acids; 53060 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: P43220 ## Belongs to the G-protein coupled receptor 2 family.
Species:
Human
Brand Family:
Chemicon
Sales Restrictions:
This product requires a User Agreement (Label License). Please see product datasheet for further information.
Protein Target:
GLP-1
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:
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 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-9 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-9 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-9 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-9 Plating Media for plating for calcium assay.
Target Sub-Family:
Glucagon
GPCR Class:
B
Promotional Text:
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Gene Symbol:
- GLP1R
- MGC138331
- GLP-1R
- GLP-1-R
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiSCREEN™ Human Recombinant GLP-1 Glucagon-Like Peptide Receptor Calcium-Optimized Stable Cell Line
Alternate Names:
GLP1R
Materials Required but Not Delivered:
Chem-9 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)
250µg/mL Genetecin/G-418
250µg/mL Hygromyocin
Chem-9 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-9 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-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)
250µg/mL Genetecin/G-418
250µg/mL Hygromyocin
Chem-9 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-9 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)
Quality Assurance:
EC50 for calcium mobilization by GLP-1: 8.4nM,
EC50 for calcium mobilization by Exendin 4: 8.5 nM,
EC50 for calcium mobilization by Exendin 4: 8.5 nM,
Cell Type:
Chem-9
Cell Line Type:
GPCR Cell Lines
Host Cells:
Chem-9
UniProt Number:
Packaging:
2 x 106 cells/vial
Entrez Gene Number: