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Human DRD1/Galpha15 Stable Cell Line-Chem-7

Cat.No.: CSC-RG0651
Cell Line Description: This human D2L-expressing cell line is made in the Chem-7 host, which supports high levels of recombinant D2 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 D2 and its ligands.
Background: Dopamine is a catecholamine neurotransmitter that functions in the CNS to control locomotor, cognitive, emotional and neurendocrine processes, and in the periphery to modulate cardiovascular, renal and gastrointestinal processes. The biological activities of dopamine are mediated by a family of five GPCRs. The D1 and D5 subtypes couple to Gs to increase intracellular cAMP, whereas the D2 , D3 and D4 subtypes couple to Gi to reduce cAMP. The D2 dopamine receptors have been of particular clinical interest due to their regulation of prolactin secretion and their affinity for antipsychotic drugs. The D2 receptor exists as two alternatively spliced isoforms differing in the insertion of a stretch of 29 amino acids in the third intracellular loop (D2S and D2L ).
Growth Properties: Adherent
Host Cell: Chem-7
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 Dopamine: ~ 76 nM; EC50 for calcium mobilization by Quinpirole: ~ 29 nM; IC50 for Raclopride with 2x EC50 Dopamine: 0.22 nM; IC50 for SCH23390 with 2x EC50 Dopamine: 831 nM.
Application: Calcium flux assay, ligand binding assays.
Sub-type: Dopamine
Propagation: Complete growth medium: F-12K Nutrient Mixture, Kaighn"s Modification + 2 mM L-glutamine + 10% heat-inactivated FBS + 100 U/mL Pen-Strep + G-418 (250ug/mL) + Zeocin (200 ug/mL) 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. Immediately upon receipt, thaw cells or place cells in liquid nitrogen. 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 Growth media. Place the flask in a humidified incubator at 37°C with 5% CO2. 3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
Subculturing: 1. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca2+ and Mg2+ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37°C with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Growth media per 1 mL trypsin. 2. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
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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