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Human C5AR1-FLAG Stable Cell Line-HEK293T

Cat.No.: CSC-RG0178
Cell Line Description: HEK293T-HuC5AR1-FLAG cell line is a hypotriploid human cell line, which has been transfected with a Human complement component 5a receptor 1 (C5AR1) tagged in the N-terminus with FLAG to allow stably express of the human C5AR1 tagged in the N-terminus with FLAG protein. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: C5a receptor (C5a anaphylatoxin chemotactic receptor, or CD88 antigen, C5R1) is a receptor for chemotactic and inflammatory peptide anaphylatoxin C5a. C5a is a classic, potent inflammatory mediator, and the C5R1 receptor stimulates chemotaxis, granule enzyme release and superoxide anion production. C5a receptor antagonists, such as AcPhe[Orn-Pro-D-cyclohexylalanine-Trp-Arg], show therapeutic effects in various inflammatory disease models. C5a receptor may also be involved in neurodegeneration and Alzheimer"s disease.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293T
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human ADORA2A.2. Protein expression: ADORA2A expression in this cell line has been validated by WB.3. Functional validation: Functional assays
Sub-type: Anaphylatoxin
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 1:10 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: C5AR1 complement component 5a receptor 1 [ Homo sapiens ]
Official Symbol: C5AR1
Synonyms: C5AR1; complement component 5a receptor 1; C5R1, complement component 5 receptor 1 (C5a ligand); C5a anaphylatoxin chemotactic receptor; C5A; C5AR; CD88; C5a-R; C5a ligand; C5a anaphylatoxin receptor; complement component 5 receptor 1; C5R1;
Gene ID: 728
mRNA Refseq: NM_001736
Protein Refseq: NP_001727
MIM: 113995
UniProt ID: P21730
Chromosome Location: 19q13.3-q13.4
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Complement and Coagulation Cascades, organism-specific biosystem; Complement and coagulation cascades, organism-specific biosystem; Complement and coagulation cascades, conserved biosystem; G alpha (i) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem;
Function: C5a anaphylatoxin receptor activity; G-protein coupled receptor activity; receptor activity; signal transducer activity;

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