READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT OX1 OREXIN FAMILY RECEPTOR
Description:
READY-TO-ASSAY™ CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT OX1 OREXIN FAMILY RECEPTOR
HUMAN RECOMBINANT OX1 OREXIN FAMILY RECEPTOR
Product Overview:
Full-length human HCRTR1 cDNA encoding OX1
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 orexin/hypocretin peptides orexin A and orexin B (also known as hcrt-1 and hcrt-2) are 33- and 28-amino acid peptides, respectively, that are preferentially expressed in hypothalamus. The orexins bind to two GPCRs, OX1 and OX2, to increase intracellular calcium. Experiments with OX1 selective antagonists have shown the receptor plays a role in food consumption and also in acquisition and consolidation of spatial memory.(Haynes et al., 2000; Akbari et al, 2006) In addition, the orexinergic system plays a role in energy metabolism and stress responses (Kukkonen et al., 2002). Millipore’s cloned OX1-expressing cell line is made in the Chem-1 host, an adherent cell line that supports high levels of recombinant OX1 expression on the cell surface and contains high levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. The untreated OX1-Chem-1 cell line and the Ready-To-Assay™ OX1 cells have equivalent EC50s for Orexin.
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 orexin/hypocretin peptides orexin A and orexin B (also known as hcrt-1 and hcrt-2) are 33- and 28-amino acid peptides, respectively, that are preferentially expressed in hypothalamus. The orexins bind to two GPCRs, OX1 and OX2, to increase intracellular calcium. Experiments with OX1 selective antagonists have shown the receptor plays a role in food consumption and also in acquisition and consolidation of spatial memory.(Haynes et al., 2000; Akbari et al, 2006) In addition, the orexinergic system plays a role in energy metabolism and stress responses (Kukkonen et al., 2002). Millipore’s cloned OX1-expressing cell line is made in the Chem-1 host, an adherent cell line that supports high levels of recombinant OX1 expression on the cell surface and contains high levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. The untreated OX1-Chem-1 cell line and the Ready-To-Assay™ OX1 cells have equivalent EC50s for Orexin.
Application Notes:
Calcium flux assay
Host Cells:
Chem-1, an adherent cell line expressing the promiscuous G-protein, Gα15
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:
- HCRTR1
- Ox1r
- OX1R
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 OX1 (nM) | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 0.5 | 3940 | 0.67 |
| Continuous Passage Cells | 1.5 | 3982 | 0.75 |
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.