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Human DRD4-FLAG Stable Cell Line-HEK293T

Cat.No.: CSC-RG0239
Cell Line Description: HEK293T-HuDRD4-FLAG cell line is a hypotriploid human cell line, which has been transfected with a Human dopamine receptor D4 (DRD4) tagged in the N-terminus with FLAG to allow stably express of the human DRD4 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: The human dopamine receptor D4 is a D2-like receptor that inhibits adenylyl cyclase activity and activates K+ channels. D4 receptor antagonists show great potential in the treatment of human personality and psychiatric disorders, such as ADHD (Attention deficit hyperactivity disorder), schizophrenia, alcoholism, and drug addiction.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293T
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human DRD4.2. Protein expression: DRD4 expression in this cell line has been validated by WB.3. Functional validation: Functional assays.
Sub-type: Dopamine
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: DRD4 dopamine receptor D4 [ Homo sapiens ]
Official Symbol: DRD4
Synonyms: DRD4; dopamine receptor D4; D dopamine receptor; dopamine D4 receptor; D(2C) dopamine receptor; seven transmembrane helix receptor; D4DR;
Gene ID: 1815
mRNA Refseq: NM_000797
Protein Refseq: NP_000788
MIM: 126452
UniProt ID: P21917
Chromosome Location: 11p15.5
Pathway: Amine ligand-binding receptors, organism-specific biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Dopamine receptors, organism-specific biosystem; Dopaminergic synapse, organism-specific biosystem; Dopaminergic synapse, conserved biosystem; G alpha (i) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem;
Function: G-protein coupled receptor activity; SH3 domain binding; dopamine binding; dopamine receptor activity; dopamine receptor activity, coupled via Gi/Go; dopamine receptor activity, coupled via Gi/Go; drug binding; potassium channel regulator activity; protein binding; receptor activity; signal transducer activity;

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