READY-TO-ASSAYTM CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT S1P4 LYSOPHOSPHOLIPID RECEPTOR
Description:
READY-TO-ASSAYTM CALCIUM-OPTIMIZED CELLS
HUMAN RECOMBINANT S1P4 LYSOPHOSPHOLIPID RECEPTOR
HUMAN RECOMBINANT S1P4 LYSOPHOSPHOLIPID RECEPTOR
Product Overview:
Full-length human EDG6 cDNA encoding S1P4
Background Information:
Millipore’s Ready-To-AssayTM 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-AssayTM cells are derived from ChemiScreenTM 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-AssayTM 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-AssayTM cells is identical to that of the originating GPCR cell line.
Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that transmits signals through a family of five G-protein-coupled receptors to regulate cell proliferation, migration, cytoskeletal organization, and differentiation (Spiegel and Milstien , 2003). S1P4 receptor was reported to couple to multiple subsets of heterotrimeric G proteins (including Gi/o, and G12/13, and possibly Gs). Unlike other S1P receptors, S1P4 expression is restricted in human and mouse to lymph node, spleen, lung, and thymus (Ishii et al., 2001). This expression pattern suggests potential roles of S1P4 in the immune system. Millipore’s cloned S1P4-expressing cell line is made in the Chem-5 host, an adherent cell line that supports high levels of recombinant S1P4 expression on the cell surface and contains high levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. The untreated S1P4-Chem-5 cell line and the Ready-To-AssayTM S1P4 cells have equivalent EC50s for S1P.
The Ready-To-AssayTM cells are derived from ChemiScreenTM 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-AssayTM 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-AssayTM cells is identical to that of the originating GPCR cell line.
Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that transmits signals through a family of five G-protein-coupled receptors to regulate cell proliferation, migration, cytoskeletal organization, and differentiation (Spiegel and Milstien , 2003). S1P4 receptor was reported to couple to multiple subsets of heterotrimeric G proteins (including Gi/o, and G12/13, and possibly Gs). Unlike other S1P receptors, S1P4 expression is restricted in human and mouse to lymph node, spleen, lung, and thymus (Ishii et al., 2001). This expression pattern suggests potential roles of S1P4 in the immune system. Millipore’s cloned S1P4-expressing cell line is made in the Chem-5 host, an adherent cell line that supports high levels of recombinant S1P4 expression on the cell surface and contains high levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. The untreated S1P4-Chem-5 cell line and the Ready-To-AssayTM S1P4 cells have equivalent EC50s for S1P.
Application Notes:
Calcium flux assay
Host Cells:
Chem-5, 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:
- EDG6
- SLP4
- LPC1
- S1P4
- S1PR4
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 (nM) | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 29.2 | 3251 | 0.79 |
| Continuous Passage Cells | 16.0 | 2584 | 0.76 |
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.