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Human DRD5-tGFP Stable Cell Line-SHSY5Y

Cat.No.: CSC-RG0038
Cell Line Description: SHSY5Y-HuDRD5-tGFP cell line is a bone marrow neuroblastoma cell line of the human brain, which has been transfected with a Human Dopaminergic Receptor D5 (DRD5) tagged in the N-terminus with tGFP to allow stably express of the human DRD5 tagged in the N-terminus with tGFP Protein. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: The gene encodes the D5 subtype of the Dopamine receptor. This subtype is a G-protein coupled receptor which stimulates adenylyl cyclase. Dopamine is one of the most important neurotransmitters and the expression of its receptors is well characterized in brain.Dopamine receptors are involved in many neurological processes so their abnormal signalling is implicated in several neuropsychiatric disorders. Defects in DRD5 are a cause of benign essential blepharospasm (BEB), a primary focal dystonia affecting the orbicularis oculi muscles.
Growth Properties: Mixed, adherent and suspension
Morphology: Epithelial
Host Cell: SHSY5Y
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human DRD5.2. Functional validation: Activation and Internalization assay for DRD5-tGFP(Ec50 =9.12 x 10^-7M)
Sub-type: Dopamine
Propagation: Complete growth medium: Eagle"s Minimum Essential Medium and F12 Medium. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.Atmosphere: 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 1.0 to 2.0 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.6. Incubate cultures at 37°C.Subcultivation Ratio: A subcultivation ratio of 1:20 to 1:50 is recommended Medium Renewal: every 4 to 7 days
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Complete growth medium 95%; DMSO, 5%
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: DRD5 dopamine receptor D5 [ Homo sapiens ]
Official Symbol: DRD5
Synonyms: DRD5; dopamine receptor D5; DRD1L2; D(1B) dopamine receptor; DRD1B; dopamine D5 receptor; dopamine receptor D1B; d dopamine receptor; D1beta dopamine receptor; DBDR; MGC10601;
Gene ID: 1816
mRNA Refseq: NM_000798
Protein Refseq: NP_000789
UniProt ID: P21918
Chromosome Location: 4p16.1
Pathway: Amine ligand-binding receptors, organism-specific biosystem; Calcium signaling pathway, organism-specific biosystem; Calcium signaling pathway, conserved biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Dopamine receptors, organism-specific biosystem; Dopaminergic synapse, organism-specific biosystem; Dopaminergic synapse, conserved biosystem;
Function: G-protein alpha-subunit binding; G-protein coupled receptor activity; dopamine binding; dopamine receptor activity; dopamine receptor activity, coupled via Gs; drug binding; receptor activity; signal transducer activity;

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