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Human DRD1/Galpha15 Stable Cell Line-Chem-6

Cat.No.: CSC-RG0650
Cell Line Description: This human D1-expressing cell line is made in the Chem-6 host, which supports high levels of recombinant D1 expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between D1 and its ligands.
Background: Dopamine exerts its action by binding to five distinct dopamine receptors, all of which belong to G protein-coupled receptor family. The D1 subtype is the most abundant dopamine receptor in the central nervous system. Activation of D1 receptor stimulates adenylyl cyclase, activates cyclic AMP-dependent protein kinases. It regulates neuronal growth and development, mediate some behavioral responses and modulate dopamine receptor D2 -mediated events.
Growth Properties: Adherent
Host Cell: Chem-6
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of DRD1. 2. Protein expression: DRD1 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by A68930: ~ 0.097 nM; Signal:noise at agonist Emax: 452.
Application: Calcium flux assay, ligand binding assays
Sub-type: Dopamine
Propagation: Complete growth medium: F-12K Nutrient Mixture, Kaighn"s Modification + 10% heat inactivated FBS + 0.25 mg/mL Geneticin(G418) + 0.2mg/mL Zeocin + 100 U/mL penicillin + 100 U/mL streptomycin.Plating medium: F-12K Nutrient Mixture, Kaighn"s Modification + 10% heat-inactivated FBS + 1x Pen-StrepAtmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37 °C
Starting Cells From Frozen Cell Stock: 1. Place cells in liquid nitrogen immediately upon receipt. Maintain frozen in liquid nitrogen for up to 5 years. 2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37°C water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing 20 mL growth media, and place in a humidified 37°C incubator with 5% CO2.
Subculturing: 1. After 8-24 h, cells will adhere to the plate, at which time the media should be replaced to remove residual DMSO. Cells are passaged by washing with Ca2+ and Mg2+ -free HBSS (10 mL/T75), incubating with 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) for 5-10 minutes at 37°C, and rapping the side of the flask to dislodge the cells. Neutralize the trypsin by addition of 4 volumes growth media. 2. Cells are typically passaged 1:10 with every 3-4 days, and should be passaged at least once after thawing prior to use in calcium flux assays.
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Freezing Medium: F-12K Nutrient Mixture, Kaighn"s Modification + 20% heat-inactivated FBS + 1x Pen-Strep + 10% DMSO
Storage: Liquid nitrogen
Preservation: 1. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Subculture-Step 1). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 10^6 cells/mL in Freezing Media (cell densities of 2-10 x 10^6 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at -70°C overnight. Store the vials in liquid nitrogen. 2. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Plating Media for plating for calcium assay.
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: DRD1 dopamine receptor D1 [ Homo sapiens ]
Official Symbol: DRD1
Synonyms: DRD1; dopamine receptor D1; D(1A) dopamine receptor; dopamine D1 receptor; DADR; DRD1A;
Gene ID: 1812
mRNA Refseq: NM_000794
Protein Refseq: NP_000785
MIM: 126449
UniProt ID: P21728
Chromosome Location: 5q34-q35
Pathway: Amine ligand-binding receptors, organism-specific biosystem; Amphetamine addiction, organism-specific biosystem; Amphetamine addiction, conserved biosystem; Calcium signaling pathway, organism-specific biosystem; Calcium signaling pathway, conserved biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Cocaine addiction, organism-specific biosystem;
Function: G-protein coupled receptor activity; dopamine binding; dopamine receptor activity; dopamine receptor activity, coupled via Gs; dopamine receptor activity, coupled via Gs; drug binding; protein binding; receptor activity; signal transducer activity;

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