ChemiSCREEN™ Human Recombinant LPA3 Lysophospholipid Receptor Calcium-Optimized Ready-to-Assay Cells
Description:
ChemiSCREEN™ Human Recombinant LPA3 Lysophospholipid Receptor Calcium-Optimized Ready-to-Assay Cells
Product Overview:
Full-length human EDG7 cDNA encoding LPA3
Background Information:
Millipore's Ready-To-Assay Calcium-Optimized Cells are GPCR-expressing cells 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.
Lysophosphatidic acid (LPA) is a lysophospholipid produced by activated platelets that inhibits adenylate cyclase and stimulates DNA synthesis, changes in cell morphology, and increases in intracellular calcium in a variety of cultured mammalian cells. A family of three GPCRs, LPA1, LPA2, and LPA3, mediates the biological effects of LPA (Contos et al., 2000). LPA3 is expressed primarily in testes, prostate, heart and pancreas. Mice lacking LPA3 have low expression of uterine COX-2 and display decreased uterine implantation and embryo spacing (Ye et al., 2005). Millipore's cloned human LPA3 -expressing cells are made in the Chem-1 host, an adherent cell line. The untreated LPA3 -Chem-1 cell line and the Ready-To-Assay LPA3 cells have equivalent EC50s for Oleoyl-L-α-lysophosphatidic acid.
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.
Lysophosphatidic acid (LPA) is a lysophospholipid produced by activated platelets that inhibits adenylate cyclase and stimulates DNA synthesis, changes in cell morphology, and increases in intracellular calcium in a variety of cultured mammalian cells. A family of three GPCRs, LPA1, LPA2, and LPA3, mediates the biological effects of LPA (Contos et al., 2000). LPA3 is expressed primarily in testes, prostate, heart and pancreas. Mice lacking LPA3 have low expression of uterine COX-2 and display decreased uterine implantation and embryo spacing (Ye et al., 2005). Millipore's cloned human LPA3 -expressing cells are made in the Chem-1 host, an adherent cell line. The untreated LPA3 -Chem-1 cell line and the Ready-To-Assay LPA3 cells have equivalent EC50s for Oleoyl-L-α-lysophosphatidic acid.
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.
Alternate Names:
EDG7
Host Cells:
Chem-1, an adherent cell line expressing the promiscuous G protein, Gα15.
Packaging:
1 x 107 cells/vial
Materials Required but Not Delivered:
DMEM containing 4.5 g/L glucose and 4 mM L-glutamine/10% heat inactivated fetal bovine serum/1x nonessential amino acids/10 mM HEPES//100 U/ml each penicillin and streptomycin
Gene Symbol:
- EDG7
- LP-A3
- LPA-3
- RP4-678I3
- Edg-7
- HOFNH30
- LPA3
- LPAR3
- GPCR
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 Oleoyl-L-α-lysophosphatidic acid (nM) | Maximum Signal (RFU) | Z' | |
| Ready-To-Assay Cells | 169 | 3241 | 0.72 |
| Continuous Passage Cells | 114 | 3954 | 0.9 |
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 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 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 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.