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Human DRD4/Galpha15 Stable Cell Line-Chem-5

Cat.No.: CSC-RG0653
Cell Line Description: This human D4-expressing cell line is made in the Chem-5 host, which supports high levels of recombinant D4 expression on the cell surface and contains optimal levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists at D4.
Background: The neurotransmitter dopamine is involved in a wide variety of neuroendocrine, emotional, cognitive and locomotor functions. These activities of dopamine are mediated by a group of 5 G protein coupled receptors, 2 of which (D1 and D5) couple to Gs and 3 of which (D2 , D3 , D4) couple to Gi. The D4 receptor is highly polymorphic, particularly in the third cytoplasmic loop, which contains from 2 to 7 repeats of 16 amino acids. Individuals with 7 repeats have increased risk of developing ADHD. Mice engineered to lack D4 have reduced locomotor activity, increased sensitivity to drugs of abuse that elevate dopamine (cocaine, methamphetamine and ethanol), and reduced response to novelty.
Growth Properties: Adherent
Host Cell: Chem-5
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of DRD4.2. Protein expression: DRD4 in this cell line has been validated by immunocytochemical staining.
Application: Calcium flux assay, ligand binding assays
Sub-type: Dopamine
Propagation: Complete growth medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x Nonessential amino acids + 10mM HEPES + 1x Pen-Strep + 250μg/mL Genetecin/G-418 + 250μg/mL Hygromycin Plating medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-Strep Atmosphere: 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: 90% heat-inactivated FBS + 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: 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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