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Human S1PR1-FLAG Stable Cell Line-CHO-K1 Galphaqi5

Cat.No.: CSC-RG0289
Cell Line Description: CHO-K1 Galphaqi5-HuS1PR1-FLAG is clonally-derived from a CHO-K1 cell line which has been transfected with a human sphingosine-1-phosphate receptor 1 (S1PR1) tagged in the N-terminus with FLAG to allow stably express of the human S1PR1 tagged in the N-terminus with FLAG. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: S1P1 (or EDG1) is a widely distributed G-protein-coupled receptor for sphingosine-1-phosphate (S1P), a blood-borne bioactive lipid. Stimulation of the S1P1 receptor triggers a Gi-linked pathway, leading to growth, survival, migration, and morphogenesis. Disruption of the S1P1 gene in mice results in embryonic lethality because of its key role within endothelial cells in regulating the coverage of blood vessels by vascular smooth muscle cells. S1P1 also mediates activation of Rac and functions as a typical chemotactic receptor.
Growth Properties: Adherent
Morphology: Epithelial-like
Host Cell: CHO-K1 Galphaqi5
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human S1P1.2. Protein expression: S1P1 expression in this cell line has been validated by WB.3. Functional validation: Functional assays.
Sub-type: Lysophospholipid
Propagation: Complete growth medium: DMEM/F12, 10% FBS, 250 μg/mL hygromycin, 10 μg/mL puromycinAtmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37.0°C
Starting Cells From Frozen Cell Stock: 1. Remove the packaging cell lines from liquid nitrogen and carry out a quick thaw. Float the cells in the 37°C water bath for 2 minutes until nearly (80%) thawed. Once cells are thawed, it is important to dilute the cells 1:10 in growth media immediately to reduce the potentially toxic effects of the DMSO preservative on the cells.2. Clean the outside of the vial with 70% ethanol before opening.3. Remove the cells from the vial and add slowly into a 15ml conical tube containing 10 ml pre-warmed media. 4. Centrifuge for 3 minutes 1000 xg to pellet cells and remove the supernatant.5. Add 14 ml of media and transfer cells to a T25 flask or a 100 mm culture dish.6. Place the cells in the 37°C incubator with 5% CO2.7. Allow incubation for 3-4 days to reach confluence. The cells will re-attach to the surface over a period of several days in culture at 37°C.
Subculturing: 1. Remove and discard culture medium.2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.5. Add appropriate aliquots of the cell suspension to new culture vessels.Incubate cultures at 37°C.Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:6 weekly is recommended Medium Renewal: 2 to 3 times per week
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Complete growth medium 90%; DMSO, 10%
Storage: Liquid nitrogen
Preservation: 1. Detach cells from culture dish according to the Sub-Culture Procedure.2. Resuspend cells at a density of 5 x 10^6 cells/mL in freeze medium.Note: A T-75 culture flask typically yields enough cells for preparing two frozen vials.3. Aliquot 1 mL cells into cryogenic vials.4. Place vials in a freezing container and store at –80 °C overnight.5. Transfer vials to liquid nitrogen for long term storage. If properly stored, cells should remain stable for years.
Safety Considerations: The following safety precautions should be observed.1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum.2. No eating, drinking or smoking while handling the stable line.3. Wash hands after handling the stable line and before leaving the lab.4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells.5. All waste should be considered hazardous.6. Dispose of all liquid waste after each experiment and treat with bleach.
Ship: Dry ice
Gene Name: S1PR1 sphingosine-1-phosphate receptor 1 [ Homo sapiens ]
Official Symbol: S1PR1
Synonyms: S1PR1; sphingosine-1-phosphate receptor 1; EDG1, endothelial differentiation, sphingolipid G protein coupled receptor, 1; sphingosine 1-phosphate receptor 1; CD363; D1S3362; edg 1; S1P receptor 1; S1P receptor Edg-1; sphingosine 1-phosphate receptor EDG1; sphingosine 1-phosphate receptor Edg-1; endothelial differentiation G-protein coupled receptor 1; endothelial differentiation, sphingolipid G-protein-coupled receptor, 1; EDG1; S1P1; ECGF1; EDG-1; CHEDG1; FLJ58121;
Gene ID: 1901
mRNA Refseq: NM_001400
Protein Refseq: NP_001391
MIM: 601974
UniProt ID: P21453
Chromosome Location: 1p21
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Fc-epsilon receptor I signaling in mast cells, organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Other, organism-specific biosystem; Lysosphingolipid and LPA receptors, organism-specific biosystem;
Function: G-protein coupled receptor activity; receptor activity; signal transducer activity; sphingolipid binding; sphingosine-1-phosphate receptor activity;

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