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Human CCR7/Galpha16 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0634
Cell Line Description: This human CCR7-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant CCR7 expression on the cell surface and contains high levels of the promiscuous G protein Galpha16 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between CCR7 and its ligands.
Background: CCR7 is a GPCR that binds to CC chemokine ligands MIP-3beta (ELC/CCL19) and 6Ckine (CCL21). These ligands are expressed in the secondary lymphoid organs, and binding to CCR7 expressed in naive T cells, B cells and dendritic cells directs migration of these cells to sites of antigen presentation. Inhibition of CCR7/ligand interactions inhibits contact sensitivity, delayed type hypersensitivity, and graft vs host disease in experimental models. In addition, CCR7 expression by breast cancer, melanoma and other malignant cells are associated with lymph node metastasis.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of CCR7. 2. Protein expression: CCR7 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by MIP-3beta: 1.5 nM; EC50 for calcium mobilization by 6CKine: 5.9 nM.
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: CCR7 chemokine (C-C motif) receptor 7 [ Homo sapiens ]
Official Symbol: CCR7
Synonyms: CCR7; chemokine (C-C motif) receptor 7; CMKBR7, EBI1; C-C chemokine receptor type 7; BLR2; CD197; CDw197; CCR-7; CC-CKR-7; C-C CKR-7; MIP-3 beta receptor; CC chemokine receptor 7; chemokine (C-C) receptor 7; Epstein-Barr virus induced gene 1; EBV-induced G protein-coupled receptor 1; EBV-induced G-protein coupled receptor 1; Epstein-Barr virus induced G-protein coupled receptor; lymphocyte-specific G protein-coupled peptide receptor; epstein-Barr virus-induced G-protein coupled receptor 1; EBI1; CMKBR7;
Gene ID: 1236
mRNA Refseq: NM_001838
Protein Refseq: NP_001829
MIM: 600242
UniProt ID: P32248
Chromosome Location: 17q12-q21.2
Pathway: 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; G alpha (i) signalling events, organism-specific biosystem;
Function: C-C chemokine receptor activity; G-protein coupled receptor activity; chemokine (C-C motif) ligand 19 binding; chemokine (C-C motif) ligand 21 binding; 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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