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Human CXCR3/Clytin Stable Cell Line-HEK293

Cat.No.: CSC-RG0641
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 human CXCR3 receptor-expressing cells were made by stable transfection of HEK293 cells with clytin, the 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 CXCR3 receptor.
Background: CXCR3 is a 7-TM GPCR that is selective for the CXC chemokines IP10, I-TAC and MIG. Binding of IP10 and MIG to CXCR3 induces Ca2+ mobilization, chemotaxis and inflammatory responses of T lymphocytes, and also act as potent inhibitors of angiogenesis. CXCR3 is highly expressed in IL-2-activated T lymphocytes in vitro, and in T lymphocytes present in inflamed tissues in rheumatoid arthritis and multiple sclerosis. In vivo, neutralization of CXCR3 inhibits experimentally induced type I diabetes, peritonitis, and post-lung transplantation bronchiolitis obliterans syndrome.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of CXCR3.2. Protein expression: CXCR3 in this cell line has been validated by immunocytochemical staining.
Application: Calcium Flux Assays
Sub-type: Chemokine
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: CXCR3 chemokine (C-X-C motif) receptor 3 [ Homo sapiens ]
Official Symbol: CXCR3
Synonyms: CXCR3; chemokine (C-X-C motif) receptor 3; G protein coupled receptor 9 , GPR9; C-X-C chemokine receptor type 3; CD183; CKR L2; CMKAR3; IP10 R; MigR; CXC-R3; CXCR-3; Mig receptor; IP10 receptor; IP-10 receptor; G protein-coupled receptor 9; chemokine (C-X-C) receptor 3; interferon-inducible protein 10 receptor; GPR9; CD182; Mig-R; CKR-L2; IP10-R;
Gene ID: 2833
mRNA Refseq: NM_001142797
Protein Refseq: NP_001136269
MIM: 300574
UniProt ID: P49682
Chromosome Location: Xq13
Pathway: CXCR3-mediated signaling events, organism-specific biosystem; Chemokine receptors bind chemokines, organism-specific biosystem; Chemokine signaling pathway, organism-specific biosystem; Chemokine signaling pathway, conserved biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Cytokine-cytokine receptor interaction, organism-specific biosystem; Cytokine-cytokine receptor interaction, conserved biosystem;
Function: C-X-C chemokine receptor activity; G-protein coupled receptor activity; chemokine binding; chemokine receptor activity; receptor activity; signal transducer activity;

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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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