READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT SECRETIN RECEPTOR, GLUCAGON RECEPTOR FAMILY
Description:
READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT SECRETIN RECEPTOR, GLUCAGON RECEPTOR FAMILY
HUMAN RECOMBINANT SECRETIN RECEPTOR, GLUCAGON RECEPTOR FAMILY
Product Overview:
Full-length human SCTR cDNA encoding the Secretin Receptor
Background Information:
Millipore’s Ready-To-Assay™ Calcium-Optimized Cells are GPCR-expressing cell lines that are designed for simple, rapid calcium assays with no requirement for culturing cells. The user simply thaws the cells with maximal viability, dispenses into assay plates, and assays for calcium response the next day.
The Ready-To-Assay™ cells are derived from ChemiScreen™ calcium-optimized stable cell lines, which express the GPCR target of interest at high levels on the cell surface, in a host cell line containing high levels of the promiscuous Gα15 protein to couple the receptor to the calcium signaling pathway. The Ready-To-Assay™ cells are prepared by chemical treatment at a concentration optimized for effective growth arrest while maintaining high viability (>80%) after thawing and overnight plating. Pharmacological functionality of the Ready-To-Assay™ cells is identical to that of the originating GPCR cell line.
Secretin, a member of the secretin-glucagon peptide hormone family, is a 27 amino acid peptide that was originally isolated from the duodenum. In the duodenum, secretin is released to stimulate the release of digestive juices in the pancreas (Bayliss et al, 1902). The receptor for secretin is a class 2 (or class B) G protein coupled receptor that signals through Gs to stimulate cAMP production (Dong et al, 2002). Along with its traditional role in the pancreas, studies in secretin-deficient mice have shown miscommunication between the CA3 Schaffer collateral and CA1 pyramidal neurons, causing a deficiency in synaptic transmission (Nishijima et al, 2006). This miscommunication of CA1 dendrites is found in Autism, Rett Syndrome, and most forms of mental retardation, suggesting secretin could be a potential target for treatment of these disorders. Millipore’s cloned human secretin -expressing cell line is made in the Chem-1 host cells, an adherent cell line that supports high levels of recombinant secretin expression on the cell surface and contains high levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. The untreated human secretin-Chem-1 cell line and the Ready-To-Assay™ human secretin cells have equivalent EC50s for secretin.
The Ready-To-Assay™ cells are derived from ChemiScreen™ calcium-optimized stable cell lines, which express the GPCR target of interest at high levels on the cell surface, in a host cell line containing high levels of the promiscuous Gα15 protein to couple the receptor to the calcium signaling pathway. The Ready-To-Assay™ cells are prepared by chemical treatment at a concentration optimized for effective growth arrest while maintaining high viability (>80%) after thawing and overnight plating. Pharmacological functionality of the Ready-To-Assay™ cells is identical to that of the originating GPCR cell line.
Secretin, a member of the secretin-glucagon peptide hormone family, is a 27 amino acid peptide that was originally isolated from the duodenum. In the duodenum, secretin is released to stimulate the release of digestive juices in the pancreas (Bayliss et al, 1902). The receptor for secretin is a class 2 (or class B) G protein coupled receptor that signals through Gs to stimulate cAMP production (Dong et al, 2002). Along with its traditional role in the pancreas, studies in secretin-deficient mice have shown miscommunication between the CA3 Schaffer collateral and CA1 pyramidal neurons, causing a deficiency in synaptic transmission (Nishijima et al, 2006). This miscommunication of CA1 dendrites is found in Autism, Rett Syndrome, and most forms of mental retardation, suggesting secretin could be a potential target for treatment of these disorders. Millipore’s cloned human secretin -expressing cell line is made in the Chem-1 host cells, an adherent cell line that supports high levels of recombinant secretin expression on the cell surface and contains high levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. The untreated human secretin-Chem-1 cell line and the Ready-To-Assay™ human secretin cells have equivalent EC50s for secretin.
Application Notes:
Calcium flux assay
Host Cells:
Chem-1, an adherent cell line expressing a promiscuous G-protein.
Packaging:
1 x 107 viable cells/mL
Materials Required but Not Delivered:
PLATING 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)
100 U/mL Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
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)
100 U/mL Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
Gene Symbol:
- SCTR
- SR
- SCT-R
Presentation:
Cells are frozen at 1 x 107 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO.
Quality Assurance:
| EC50 for secretin (nM) | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 3.5 | 6852 | 0.62 |
| Continuous Passage Cells | 5.9 | 7664 | 0.62 |
Storage Conditions:
Place cells in liquid nitrogen immediately upon receipt. Maintain frozen in liquid nitrogen for up to 5 years.
1) 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.
2) Transfer contents of the vial to a sterile 15 mL conical tube. Add 10 mL prewarmed plating media to the cells and mix gently to resuspend cells. Centrifuge at 200 x g. Remove all but 0.5 mL media.
3) Resuspend cells to 0.5 x 106 cells/mL in plating media. Dispense the cell suspension into a 96-well assay plate at 200 µL per well to obtain a density of approximately 1 x 105 cells/well.
4) Place the assay plate in a humidified 37°C incubator with 5% CO2.
5) The cells may be assayed 16-24 hours after plating. It is recommended to wash the cells with assay buffer at least once prior to addition of loading dye.
1) 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.
2) Transfer contents of the vial to a sterile 15 mL conical tube. Add 10 mL prewarmed plating media to the cells and mix gently to resuspend cells. Centrifuge at 200 x g. Remove all but 0.5 mL media.
3) Resuspend cells to 0.5 x 106 cells/mL in plating media. Dispense the cell suspension into a 96-well assay plate at 200 µL per well to obtain a density of approximately 1 x 105 cells/well.
4) Place the assay plate in a humidified 37°C incubator with 5% CO2.
5) The cells may be assayed 16-24 hours after plating. It is recommended to wash the cells with assay buffer at least once prior to addition of loading dye.