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Human CXCR4/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0642
Cell Line Description: This human recombinant CXCR4-expresssing cell lines is made in Chem-1 host, which support high levels of recombinant CXCR4 expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between CXCR4 and its ligands.
Background: The chemokine SDF-1alpha and its GPCR receptor CXCR4 have a one-to-one specificity that is unique among chemokines and their receptors. SDF-1alpha binds to CXCR4 expressed on hematopoietic and lymphopoietic cells, and directs their trafficking to and retention in hemato-and lymphatopoietic organs and sites of inflammation. CXCR4 is expressed on several tumor cell lineages, and might be responsible for metastasis to sites of SDF-1alpha expression, such as bone and lymph nodes. In addition, CXCR4 is a coreceptor for the HIV envelope glycoprotein gp120. Small molecule antagonists of CXCR4 have been developed and shown to inhibit infectivity of T-tropic HIV strains and to impair growth of brain tumors.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of CXCR4.2. Protein expression: CXCR4 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by SDF-1alpha: ~ 4 nM; Signal/noise at ligand Emax: 542; Z = 0.82 with SDF-1alpha at EC50.
Application: Calcium flux assay, ligand binding assays.
Sub-type: Chemokine
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: CXCR4 chemokine (C-X-C motif) receptor 4 [ Homo sapiens ]
Official Symbol: CXCR4
Synonyms: CXCR4; chemokine (C-X-C motif) receptor 4; chemokine (C X C motif), receptor 4 (fusin); C-X-C chemokine receptor type 4; CD184; D2S201E; fusin; HM89; HSY3RR; LESTR; NPY3R; NPYR; NPYY3R; CXC-R4; CXCR-4; CD184 antigen; SDF-1 receptor; neuropeptide Y receptor Y3; seven transmembrane helix receptor; stromal cell-derived factor 1 receptor; lipopolysaccharide-associated protein 3; seven-transmembrane-segment receptor, spleen; leukocyte-derived seven transmembrane domain receptor; leukocyte-derived seven-transmembrane-domain receptor; FB22; LAP3; LCR1; WHIM; NPYRL;
Gene ID: 7852
mRNA Refseq: NM_001008540
Protein Refseq: NP_001008540
MIM: 162643
UniProt ID: P61073
Chromosome Location: 2q21
Pathway: Axon guidance, organism-specific biosystem; Axon guidance, conserved biosystem; Binding and entry of HIV virion, organism-specific biosystem; CXCR4-mediated signaling events, organism-specific biosystem; Chemokine receptors bind chemokines, organism-specific biosystem; Chemokine signaling pathway, organism-specific biosystem; Chemokine signaling pathway, conserved biosystem;
Function: C-X-C chemokine receptor activity; G-protein coupled receptor activity; actin binding; coreceptor activity; myosin light chain binding; protein binding; receptor activity; signal transducer activity; ubiquitin binding; ubiquitin protein ligase binding;

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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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