ChemiSCREEN™ Human Recombinant S1P2 Lysophospholipid Receptor Calcium-Optimized Ready-to-Assay ™ Cells
Description:
ChemiSCREEN™ Human Recombinant S1P2 Lysophospholipid Receptor Calcium-Optimized Ready-to-Assay ™ Cells
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.
Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to and activates a family of GPCRs, S1P1-5 (also known as EDG receptors). Interactions between S1P and its receptors mediate cytoskeletal rearrangement and cell migration, with functional consequences in angiogenesis, lymphocyte trafficking, and smooth muscle development (Anliker and Chun, 2004). S1P1 (Edg-1) signals exclusively through Gi, whereas S1P2 (Edg-5) and S1P3 (Edg-3) activate Gi, Gq and G12/13 (Windh et al., 1999). Although S1P1 and S1P3 promote cell migration, S1P2 inhibits cell migration in several cell types; these opposing functions appear to result from differences in the ability of each receptor to activate Gi (Arikawa et al., 2003; Sugimoto et al., 2003; Goparaju et al., 2005). Studies with knockout mice indicate that S1P2 and S1P3 have redundant functions in maintaining vascular integrity during embryonic development (Kono et al., 2004). Millipore's cloned human S1P2-expressing cells are made in the Chem-1 host, an adherent cell line. The untreated S1P2-Chem-1 cell line and the Ready-To-Assay S1P2 cells have equivalent EC50s for S1P.
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.
Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to and activates a family of GPCRs, S1P1-5 (also known as EDG receptors). Interactions between S1P and its receptors mediate cytoskeletal rearrangement and cell migration, with functional consequences in angiogenesis, lymphocyte trafficking, and smooth muscle development (Anliker and Chun, 2004). S1P1 (Edg-1) signals exclusively through Gi, whereas S1P2 (Edg-5) and S1P3 (Edg-3) activate Gi, Gq and G12/13 (Windh et al., 1999). Although S1P1 and S1P3 promote cell migration, S1P2 inhibits cell migration in several cell types; these opposing functions appear to result from differences in the ability of each receptor to activate Gi (Arikawa et al., 2003; Sugimoto et al., 2003; Goparaju et al., 2005). Studies with knockout mice indicate that S1P2 and S1P3 have redundant functions in maintaining vascular integrity during embryonic development (Kono et al., 2004). Millipore's cloned human S1P2-expressing cells are made in the Chem-1 host, an adherent cell line. The untreated S1P2-Chem-1 cell line and the Ready-To-Assay S1P2 cells have equivalent EC50s for S1P.
Application Notes:
Calcium flux assay
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.
Host Cells:
Chem-1, an adherent cell line expressing the promiscuous G-protein, Gα15
Packaging:
1 x 107 cells/vial
Gene Symbol:
- EDG5
- S1P2
- Gpcr13
- AGR16
- EDG-5
- LPB2
- H218
- S1PR2
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 S1P | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 6.74 nM | 4788 | 0.67 |
| Continuous Passage Cells | 16.67 nM | 7561 | 0.54 |
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 37°C 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 Chem-1 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 Chem-1 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 37°C 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 Chem-1 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 Chem-1 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.