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Human S1PR4-FLAG Stable Cell Line-CHO-K1 Galphaqi5

Cat.No.: CSC-RG0292
Cell Line Description: CHO-K1 Galphaqi5-HuS1PR4-FLAG is clonally-derived from a CHO-K1 cell line which has been transfected with a human sphingosine-1-phosphate receptor 4 (S1PR4) tagged in the N-terminus with FLAG to allow stably express of the human S1PR4 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: S1P4 receptor or Endothelial Differentiation Gene-6 (EDG-6) is receptor for sphingosine-1-phosphate (S1P). S1P is a bioactive lipid produced from the metabolism of sphingomyelin. It is an important constituent of serum and regulates cell growth, survival, migration, differentiation and gene expression. Unlike other members of S1P receptor, which are widely expressed, S1P4 exhibits lymphoid tissue-specific expression. EDG-6 has been implicated in regulation of cell shape and motility.
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 S1P4.2. Protein expression: S1P4 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: S1PR4 sphingosine-1-phosphate receptor 4 [ Homo sapiens ]
Official Symbol: S1PR4
Synonyms: S1PR4; sphingosine-1-phosphate receptor 4; EDG6, endothelial differentiation, G protein coupled receptor 6 , endothelial differentiation, lysophosphatidic acid G protein coupled receptor, 6; sphingosine 1-phosphate receptor 4; S1P receptor 4; S1P receptor Edg-6; sphingosine 1-phosphate receptor Edg-6; endothelial differentiation G-protein coupled receptor 6; endothelial differentiation, G protein coupled receptor 6; endothelial differentiation, G-protein-coupled receptor 6; endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 6; EDG6; LPC1; S1P4; SLP4;
Gene ID: 8698
mRNA Refseq: NM_003775
Protein Refseq: NP_003766
MIM: 603751
UniProt ID: O95977
Chromosome Location: 19p13.3
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; Lysosphingolipid and LPA receptors, organism-specific biosystem; Neuroactive ligand-receptor interaction, organism-specific biosystem; Neuroactive ligand-receptor interaction, conserved biosystem;
Function: G-protein coupled receptor activity; lipid binding; receptor activity; signal transducer activity; sphingosine-1-phosphate receptor activity;

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