READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT β1 ADRENERGIC RECEPTOR
Description:
READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT β1 ADRENERGIC RECEPTOR
HUMAN RECOMBINANT β1 ADRENERGIC RECEPTOR
Product Overview:
Full-length human ADRB1 cDNA encoding β1
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.
The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the α- and β-adrenergic receptors (Bylund et al., 1994). The three members of the β-adrenergic receptor family, β1, β2 and β3, couple to Gs to increase cAMP upon activation. In the heart, the β1 receptor constitutes 70-80% of the β-adrenergic receptors. Activation of cardiac β-adrenergic receptors, acutely increases heart rate, cardiac output, and cardiac automaticity, and chronically increases cardiac myocyte apoptosis. In failing hearts, the β1 subtype is downregulated and desenstitized, probably as a result of increased catecholamine levels. As a result, β-adrenergic receptor antagonists (β blockers) are effective in the treatment of congestive heart failure and arrhythmia (Lohse et al., 2003). Millipore’s cloned human β1-expressing cell line is made in the Chem-1 host cells, an adherent cell line that supports high levels of recombinant β1 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 β1-Chem-1 cell line and the Ready-To-Assay™ human β1 cells have equivalent EC50s for denopamine.
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.
The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the α- and β-adrenergic receptors (Bylund et al., 1994). The three members of the β-adrenergic receptor family, β1, β2 and β3, couple to Gs to increase cAMP upon activation. In the heart, the β1 receptor constitutes 70-80% of the β-adrenergic receptors. Activation of cardiac β-adrenergic receptors, acutely increases heart rate, cardiac output, and cardiac automaticity, and chronically increases cardiac myocyte apoptosis. In failing hearts, the β1 subtype is downregulated and desenstitized, probably as a result of increased catecholamine levels. As a result, β-adrenergic receptor antagonists (β blockers) are effective in the treatment of congestive heart failure and arrhythmia (Lohse et al., 2003). Millipore’s cloned human β1-expressing cell line is made in the Chem-1 host cells, an adherent cell line that supports high levels of recombinant β1 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 β1-Chem-1 cell line and the Ready-To-Assay™ human β1 cells have equivalent EC50s for denopamine.
Application Notes:
Calcium flux assay
Species Reactivity:
Human
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:
- ADRB1
- ADRB1R
- RHR
- B1AR
- BETA1AR
Analytes Available:
β1
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 Denopamine (nM) | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 13.4 | 6153 | 0.75 |
| Continuous Passage Cells | 13.6 | 6992 | 0.79 |
Detection Methods:
Radioactive
Kit or Assay Type:
Cell Based Assays
Protein or Enzyme Type:
GPCRs
Species:
Human
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.