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Human LTB4R-FLAG Stable Cell Line-HEK293T

Cat.No.: CSC-RG0445
Cell Line Description: HEK293T-HuLTB4R-FLAG cell line is a hypotriploid human cell line, which has been transfected with a human leukotriene B4 receptor (LTB4R) tagged in the N-terminus with FLAG to allow stably express of the human LTB4R 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: Leukotriene B4 (LTB4) is a potent lipid mediator of allergic and inflammatory reactions, as well as a modulator of immune responses. Two types of plasma membrane receptors for LTB4 have been described on human neutrophils. The high-affinity human leukocyte LTB4 receptor, BLT1 (or LTB4R) mediates aggregation, chemotaxis, chemokinesis, and increased adherence to surfaces, whereas the low-affinity receptor mediates degranulation and increased oxidative metabolism. BLT1 mRNA is expressed in leukocytes and to a lesser degree in spleen and thymus. Studies in mice with disrupted BLT1 gene indicate a major role for BLT1 in acute inflammation and immediate hypersensitivity, as well as in leukocyte functions such as chemotaxis and firm adhesion to endothelium in response to LTB4.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293T
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human LTB4R.2. Protein expression: LTB4R expression in this cell line has been validated by WB.3. Functional validation: Functional assays.
Sub-type: Leukotriene
Propagation: Complete growth medium: DMEM, 10% FBS, 1 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 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: LTB4R leukotriene B4 receptor [ Homo sapiens ]
Official Symbol: LTB4R
Synonyms: LTB4R; leukotriene B4 receptor; CMKRL1, GPR16, P2RY7; leukotriene B4 receptor 1; BLTR; LTB4R1; P2Y7; LTB4-R1; LTB4-R 1; P2Y purinoceptor 7; chemokine receptor-like 1; G protein-coupled receptor 16; G-protein coupled receptor 16; chemoattractant receptor-like 1; purinergic receptor P2Y, G-protein coupled, 7; BLT1; GPR16; LTBR1; P2RY7; CMKRL1;
Gene ID: 1241
mRNA Refseq: NM_001143919
Protein Refseq: NP_001137391
MIM: 601531
UniProt ID: Q15722
Chromosome Location: 14q11.2-q12
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Eicosanoid ligand-binding receptors, organism-specific biosystem; G alpha (q) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class A Rhodopsin-like, organism-specific biosystem; Leukotriene receptors, organism-specific biosystem;
Function: leukotriene B4 receptor activity; leukotriene receptor activity; nucleotide binding; receptor activity; signal transducer activity;

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Optional requirements on this protein    +Expand
C-fusion    N-fusion   Non-tagged
His    GST   Fc   Others
<1.0 eu/μg    <0.1 eu/μg   <0.01 eu/μg   Not required
Monomer Isolation    Dimer Isolation    Not required
>80% by SDS-PAGE    >90% by SDS-PAGE   >95% by SDS-PAGE   Others

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