ChemiSCREEN™ Human Recombinant CXCR3 Chemokine Receptor Calcium-Optimized Stable Cell Line

ChemiSCREEN™ Human Recombinant CXCR3 Chemokine Receptor Calcium-Optimized Stable Cell Line

Chemicon (Millipore)

2 vials

Full-length human CXCR3 cDNA

CXCR3 is a 7-TM GPCR that is selective for the CXC chemokines IP10, I-TAC and MIG (Loetscher et al., 1996). 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 (Loetscher et al., 1996), and in T lymphocytes present in inflamed tissues in rheumatoid arthritis and multiple sclerosis (Balashoy et al., 1999; Qin et al., 1998). In vivo, neutralization of CXCR3 inhibits experimentally induced type I diabetes (Frigerio et al., 2002), peritonitis (Xie et al., 2003), and post-lung transplantation bronchiolitis obliterans syndrome (Belperio et al., 2002). Chemicon’s cloned human CXCR3-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant CXCR3 expression on the cell surface and contains high levels of the promiscuous G protein G16 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between CXCR3 and its ligands.

Calcium flux assay, ligand binding assays

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

CD183 is a G protein-coupled receptor with selectivity for three chemokines, termed IP10 (interferon-g-inducible 10 kDa protein), Mig (monokine induced by interferon-g) and I-TAC (interferon-inducible T cell a-chemoattractant). IP10, Mig and I-TAC belong to the structural subfamily of CXC chemokines, in which a single amino acid residue separates the first two of four highly conserved Cys residues. Historically, CD183 is the third CXC chemokine receptor discovered and, therefore, commonly designated as CXCR3. Binding of chemokines to CD183 induces cellular responses that are involved in leukocyte traffic, most notably integrin activation, cytoskeletal changes and chemotactic migration. Inhibition by Bordetella pertussis toxin suggests that heterotrimeric G protein of the Gi-subclass couple to CD183. Signal transduction has not been further analyzed but may include the same enzymes that were identified in the signaling cascade induced by other chemokine receptors. As a consequence of chemokine-induced cellular desensitization (phosphorylation-dependent receptor internalization), cellular responses are typically rapid and short in duration. Cellular responsiveness is restored after dephosphorylation of intracellular receptors and subsequent recycling to the cell surface. A hallmark of CD183 is its prominent expression in in vitro cultured effector/memory T cells, and in T cells present in many types of inflamed tissues. In addition, IP10, Mig and I-TAC are commonly produced by local cells in inflammatory lesion, suggesting that CD183 and its chemokines participate in the recruitment of inflammatory cells. Therefore, CD183 is a target for the development of small molecular weight antagonists, which may be used in the treatment of diverse inflammatory diseases.

FUNCTION: SwissProt: P49682 # Receptor for CXCL9, CXCL10 and CXCL11 and mediates the proliferation of human mesangial cells (HMC). Isoform 2 is a receptor for CXCL4 and also mediates the inhibitory activities of CXCL9, CXCL10 and CXCL11 on the growth of human microvascular endothelial cells (HMVEC). Isoform 2 may play a role in angiogenesis. Isoform 3 mediates activity of CXCL11.
SIZE: 368 amino acids; 40660 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Isoform 1 and isoform 2 are mainly expressed in heart, kidney, liver and skeletal muscle. Isoform 1 is also expressed in placenta.
SIMILARITY: SwissProt: P49682 ## Belongs to the G-protein coupled receptor 1 family.

Human

Chemicon

CXCR3

Cells are frozen at 2 x 10⁶ cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.

Note: This CXCR3 cell line in the Chem-1 background replaces the CXCR3-Chem-2 cell line previously offered under this catalog number

peptide

Chemokine

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 37ºC 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 37ºC 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.

4.When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca⁺⁺ and Mg⁺⁺ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37ºC 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 Chem-1 Growth Media per 1 mL trypsin.

5.Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.

6.Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 10⁶ cells/mL in Chem-1 Freezing Media (cell densities of 2-10 x 10⁶ 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.

7.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 Chem-1 Plating Media for plating for calcium assay.

A

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• CXCR3
• CD182
• CXC-R3
• IP10-R
• IP10
• Mig-R
• CKR-L2
• CD183
• CMKAR3
• GPR9
• CXCR-3
• MigR

ChemiSCREEN™ Human Recombinant CXCR3 Chemokine Receptor Calcium-Optimized Stable Cell Line

Chem-1 Growth Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020 or equivalent)
10% heat-inactivated FBS
1x Nonessential amino acids (from 100x stock, Millipore TMS-001-C)
10mM HEPES (from 1 M HEPES, Millipore TMS-003-C)
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
G-418 (250ug/mL)

Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep

Chem-1 Freezing Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)

EC50 for calcium mobilization by IP-10: 3.4 nM
EC50 for calcium mobilization by I-TAC: 3.1 nM

Chem-1

P49682

2 x 10⁶ cells/vial

NM_001504.1