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Figure 1. Calcium flux in S1P-5-expressing Chem-5 cell line induced by S1P. S1P5-expressing Chem-5 cells and Wild-Type Chem-5 cells were ...
ChemiSCREEN™ Human Recombinant S1P5 Lysophospholipid Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant S1P5 Lysophospholipid Receptor Calcium-Optimized Stable Cell Line
Trade Name:
Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Full-length human EDG8 cDNA encoding S1P5
Background Information:
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).
S1P5 can couple with Gi/o and G12/13, and it mediates S1P induced adenylate cyclase inhibition and Ca2+ mobilization like the other S1P receptors. However, unlike the other S1P receptors, it mediates inhibition of MAPK activation and cell proliferation (Im et al., 2000). S1P5 is predominantly expressed in the white matter tracts and oligodendrocytes and is particularly abundant in the anterior commissure, corpus collosum, and optic tract (Terai et al., 2003). S1P induces process retraction in pre-oligodendrocytes and supports cell survival in mature oligodendrocytes by activating S1P5, which indicates a role for S1P5 in maturation and myelination of oligodendrocytes (Jaillard et al., 2005). Millipore's cloned human S1P5-expressing cell line is made in the Chem-5 host, which supports high levels of recombinant S1P5 expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between S1P5 and its ligands.
S1P5 can couple with Gi/o and G12/13, and it mediates S1P induced adenylate cyclase inhibition and Ca2+ mobilization like the other S1P receptors. However, unlike the other S1P receptors, it mediates inhibition of MAPK activation and cell proliferation (Im et al., 2000). S1P5 is predominantly expressed in the white matter tracts and oligodendrocytes and is particularly abundant in the anterior commissure, corpus collosum, and optic tract (Terai et al., 2003). S1P induces process retraction in pre-oligodendrocytes and supports cell survival in mature oligodendrocytes by activating S1P5, which indicates a role for S1P5 in maturation and myelination of oligodendrocytes (Jaillard et al., 2005). Millipore's cloned human S1P5-expressing cell line is made in the Chem-5 host, which supports high levels of recombinant S1P5 expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between S1P5 and its ligands.
Application Notes:
Calcium flux assay, ligand binding assays
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.
Entrez Gene Summary:
The lysosphingolipid sphingosine 1-phosphate (S1P) regulates cell proliferation, apoptosis, motility, and neurite retraction. Its actions may be both intracellular as a second messenger and extracellular as a receptor ligand. S1P and the structurally related lysolipid mediator lysophosphatidic acid (LPA) signal cells through a set of G protein-coupled receptors known as EDG receptors. Some EDG receptors (e.g., EDG1; MIM 601974) are S1P receptors; others (e.g., EDG2; MIM 602282) are LPA receptors.[supplied by OMIM]
UniProt Summary:
FUNCTION: SwissProt: Q9H228 # Receptor for the lysosphingolipid sphingosine 1- phosphate (S1P). S1P is a bioactive lysophospholipid that elicits diverse physiological effect on most types of cells and tissues. Is coupled to both the G(i/0)alpha and G(12) subclass of heteromeric G-proteins (By similarity). May play a regulatory role in the transformation of radial glial cells into astrocytes and may affect proliferative activity of these cells.
SIZE: 398 amino acids; 41775 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Widely expressed in the brain, most prominently in the corpus callosum, which is predominantly white matter. Detected in spleen, peripheral blood leukocytes, placenta, lung, aorta, and fetal spleen. Low-level signal detected in many tissue extracts. Isoform 1 is predominently expressed in peripheral tissues, whereas isoform 2 is expressed in brain, spleen and peripheral blood leucocytes.DEVELOPMENTAL STAGE: At 24 weeks of gestation, fragments of radial glial fibers are positive within the cortical plate and subplate of allocortical areas. These positive fragments often appear enlarged as varicosities and some of them terminate at blood vessels. Between 28 and 30 weeks of gestation, all iso- and allocortical areas contain immunolabelled radial glial fibers revealing curvature next to sulci. After 32 weeks of gestation, radial glial fibers gradually disappear; instead positive transitional stages between radial glia and astrocytes were found.DISEASE:SwissProt: Q9H228 # Overexpressed in leukemic large granular lymphocyte (LGL). LGL is a lymphopropliferative disorder often associated with autoimmune disease.
SIMILARITY: SwissProt: Q9H228 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 398 amino acids; 41775 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Widely expressed in the brain, most prominently in the corpus callosum, which is predominantly white matter. Detected in spleen, peripheral blood leukocytes, placenta, lung, aorta, and fetal spleen. Low-level signal detected in many tissue extracts. Isoform 1 is predominently expressed in peripheral tissues, whereas isoform 2 is expressed in brain, spleen and peripheral blood leucocytes.DEVELOPMENTAL STAGE: At 24 weeks of gestation, fragments of radial glial fibers are positive within the cortical plate and subplate of allocortical areas. These positive fragments often appear enlarged as varicosities and some of them terminate at blood vessels. Between 28 and 30 weeks of gestation, all iso- and allocortical areas contain immunolabelled radial glial fibers revealing curvature next to sulci. After 32 weeks of gestation, radial glial fibers gradually disappear; instead positive transitional stages between radial glia and astrocytes were found.DISEASE:SwissProt: Q9H228 # Overexpressed in leukemic large granular lymphocyte (LGL). LGL is a lymphopropliferative disorder often associated with autoimmune disease.
SIMILARITY: SwissProt: Q9H228 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Quality Assurance:
EC50 for calcium mobilization by S1P: ~ 74.2 nM
Cell Type:
Chem-5
Cell Line Type:
GPCR Cell Lines
Brand Family:
Chemicon
Host Cells:
Chem-5
Presentation:
Cells are frozen at 2 x 106 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.
Protein Target:
S1P5 / EDG8
UniProt Number:
Ligand Type:
non-peptide
Storage Conditions:
1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen. Maintain frozen in liquid nitrogen for up to 5 years.
2. 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. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37°C with 5% CO2.
3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
4. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca++ and Mg++ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37°C with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Chem-5 Growth Media per 1 mL trypsin.
5. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
6. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 106 cells/mL in Chem-5 Freezing Media (cell densities of 2-10 x 106 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at -70°C overnight. Store the vials in liquid nitrogen.
7. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Chem-5 Plating Media for plating for calcium assay.
2. 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. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37°C with 5% CO2.
3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
4. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca++ and Mg++ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37°C with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Chem-5 Growth Media per 1 mL trypsin.
5. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
6. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 106 cells/mL in Chem-5 Freezing Media (cell densities of 2-10 x 106 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at -70°C overnight. Store the vials in liquid nitrogen.
7. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Chem-5 Plating Media for plating for calcium assay.
Target Sub-Family:
Lysophospholipid
Promotional Text:
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GPCR Class:
A
Packaging:
2 x 106 cells/vial
Gene Symbol:
- EDG8
- SPPR-2
- SPPR-1
- Edg-8
- S1P5
- S1PR5
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiSCREEN™ Human Recombinant S1P5 Lysophospholipid Receptor Calcium-Optimized Stable Cell Line
Entrez Gene Number:
Materials Required but Not Delivered:
Chem-5 Growth 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)
250 μg/mL Genetecin/G-418
250 μg/mL Hygromycin
Chem-5 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
Chem-5 Freezing Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)
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)
250 μg/mL Genetecin/G-418
250 μg/mL Hygromycin
Chem-5 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
Chem-5 Freezing Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)