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Human TBXA2R/Clytin Stable Cell Line-HEK293

Cat.No.: CSC-RG0747
Cell Line Description: These cells express a novel variant of clytin, a calcium-activated photoprotein, to enable sensitive luminescent detection of ligand-induced calcium flux. The clytin contains a mutation that increases its affinity for calcium to a level that permits detection of cytosolic calcium in many cells with greater sensitivity than other photoproteins targeted to the mitochondria. Luminescent calcium assays offer several advantages over fluorescent calcium assays including increased sensitivity and lack of interference from fluorescent compounds. These cells are made by stable transfection of HEK293 cells with clytin, TP receptor, and a promiscuous G protein to couple the receptor to the calcium signaling pathway. These stability tested cells are ready for luminescent analysis of agonists, antagonists and modulators at the TP receptor.
Background: TP receptors (Thromboxane A2 receptors) are widely distributed among different organ systems and have been localized on both cell membranes and intracellular structures. TP receptors belong to the prostanoid G protein-coupled receptor family. Activation of TP receptors induces platelet aggregation, vascular and respiratory smooth muscle constriction, and enhances mitogenic responses of vascular smooth muscle cells that are stimulated by growth factors. The human TP receptor is encoded by a single gene that is alternatively spliced at the carboxyl terminus, resulting in two isoforms, TP (343 residues) and TP (407 residues). Both isoforms couple to Gq pathway, but couple oppositely to adenylate cyclase. The cDNA encoding human TP has been stably expressed in these TP receptor-expressing cells.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of TBXA2R.2. Protein expression: TBXA2R in this cell line has been validated by immunocytochemical staining.
Application: Calcium Flux and cAMP Assays
Sub-type: Prostanoid
Propagation: Complete growth medium: DMEM/F12 with 2.5 mM glutamine, 10% heat-inactivated FBS, 1x Nonessential amino acids, 1x Pen-Strep, 250μg/mL Genetecin/G-418Plating medium: DMEM/F12 with 2.5 mM glutamine, 10% heat-inactivated FBS, 1x Nonessential amino acids, 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 Accutase at 1 mL/T75 and keep at room temperature until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize Accutase by addition of 4 mL Growth Media per 1 mL Accutase. 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: TBXA2R thromboxane A2 receptor [ Homo sapiens ]
Official Symbol: TBXA2R
Synonyms: TBXA2R; thromboxane A2 receptor; prostanoid TP receptor; TXA2-R; BDPLT13;
Gene ID: 6915
mRNA Refseq: NM_001060
Protein Refseq: NP_001051
MIM: 188070
UniProt ID: P21731
Chromosome Location: 19p13.3
Pathway: Calcium signaling pathway, organism-specific biosystem; Calcium signaling pathway, conserved biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Eicosanoid ligand-binding receptors, organism-specific biosystem; G alpha (12/13) signalling events, organism-specific biosystem; G alpha (q) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem;
Function: G-protein coupled receptor activity; guanyl-nucleotide exchange factor activity; protein binding; receptor activity; signal transducer activity; thromboxane A2 receptor 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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