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Human BRS3/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0620
Cell Line Description: This human BB3-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant BB3 expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to enhance coupling of the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists at BB3.
Background: Bombesin, a bioactive peptide first identified in amphibian skin, is related to two mammalian peptides, gastrin-releasing peptide (GRP) and neuromedin B. A family of 3 GPCRs, including GRP-R (BB1), NMB-R (BB2) and BRS-3 (BB3), mediate the biological effects of the peptides. BB3 differs from the others by its low affinity for bombesin. Although an endogenous ligand for BB3 has yet to be identified, a synthetic nonselective bombesin-like peptide [D-Phe6, beta-Ala11, Phe13, Nle14]-bombesin-(6-14)-amide activates BB3 with high potency. BB3-null mice develop mild obesity that can be alleviated by treatment with an antiobesity compound, the serotonin and noradrenaline reuptake inhibitor Sibutramine.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of BRS3.2. Protein expression: BRS3 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by [D-Phe6, beta-Ala6, Phe13, Nle14]-Bombesin-(6-14)-amide: ~ 1.35 nM EC50 for calcium mobilization by Bombesin: >100 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Bombesin
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 Plating medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 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: DMEM with 4.5 g/L glucose + 4mM glutamine + 20% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 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: BRS3 bombesin-like receptor 3 [ Homo sapiens ]
Official Symbol: BRS3
Synonyms: BRS3; bombesin-like receptor 3; bombesin receptor subtype-3; BRS-3; G-protein coupled receptor; bombesin receptor subtype 3;
Gene ID: 680
mRNA Refseq: NM_001727
Protein Refseq: NP_001718
MIM: 300107
UniProt ID: P32247
Chromosome Location: Xq26.3
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (q) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class A Rhodopsin-like, organism-specific biosystem; Neuroactive ligand-receptor interaction, organism-specific biosystem; Neuroactive ligand-receptor interaction, conserved biosystem;
Function: G-protein coupled receptor activity; bombesin receptor activity; receptor activity; signal transducer activity;

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Optional requirements on this protein    +Expand
C-fusion    N-fusion   Non-tagged
His    GST   Fc   Others
<1.0 eu/μg    <0.1 eu/μg   <0.01 eu/μg   Not required
Monomer Isolation    Dimer Isolation    Not required
>80% by SDS-PAGE    >90% by SDS-PAGE   >95% by SDS-PAGE   Others

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