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Human EDNRB/Galpha15 Stable Cell Line-CHO

Cat.No.: CSC-RG0656
Cell Line Description: This human ETB-expressing cell line is made in the CHO host, which supports high levels of recombinant ETB expression on the cell surface. Thus, the cell line is an ideal tool for screening for antagonists of interactions between ETB and its ligands.
Background: The endothelin family of peptides are potent vasoconstrictors synthesized by endothelial cells in both constitutive and inducible pathways. The biological actions of endothelins are mediated by two GPCRs, ETA and ETB. Whereas ETA is the predominant receptor on smooth muscle cells and thus is the primary mediator of the vasoconstrictor activity, ETB is found on endothelial cells and mediates vasodilation. In addition, ETB has also been linked to renal failure, congestive heart failure, atherosclerosis, and pulmonary hypertension. A mutation in ETB has been found to cause Hirschsprung disease-2, a degenerative disease of the digestive tract.
Growth Properties: Adherent
Morphology: Epithelial-like
Host Cell: CHO
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of EDNRB.2. Protein expression: EDNRB in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by Endothelin-1: ~ 0.161 nM; EC50 for calcium mobilization by Endothelin-3: ~ 0.62 nM.
Application: calcium flux assay and ligand binding assay
Sub-type: Endothelin
Propagation: Complete growth medium: F-12K Nutrient Mixture, Kaighn"s Modification + 10% dialyzed heat inactivated FBS + 0.25 mg/mL Geneticin (G418) + 100 U/mL penicillin + 100 U/mL streptomycin.Plating medium: F12-K containing 2 mM L-glutamine, 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. 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 CHO 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: EDNRB endothelin receptor type B [ Homo sapiens ]
Official Symbol: EDNRB
Synonyms: EDNRB; endothelin receptor type B; HSCR, HSCR2; endothelin B receptor; ETB; endothelin receptor non-selective type; ET-B; ETBR; ETRB; HSCR; WS4A; ABCDS; ET-BR; HSCR2;
Gene ID: 1910
mRNA Refseq: NM_000115
Protein Refseq: NP_000106
MIM: 131244
UniProt ID: P24530
Chromosome Location: 13q22
Pathway: Arf6 trafficking events, organism-specific biosystem; Calcium signaling pathway, organism-specific biosystem; Calcium signaling pathway, conserved biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Endothelins, organism-specific biosystem; G alpha (q) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem;
Function: G-protein coupled receptor activity; endothelin receptor activity; endothelin receptor activity; endothelin receptor activity; peptide hormone binding; receptor activity; signal transducer activity;

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