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Figure 1. Calcium flux in CCR7-expressing Chem-1 cell line induced by MIP-3β. CCR7-
expressing Chem-1 cells and wild-type (WT) Chem-1 cells (HTS...
ChemiSCREEN™ CCR7 Calcium-Optimized FLIPR Cell Line
Description:
ChemiSCREEN™ CCR7 Calcium-Optimized FLIPR Cell Line
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
TRANSFECTION: Full-length human CCR7 cDNA
Note: This CCR7 cell line in the Chem-1 background replaces the CCR7-Chem-2 cell line previously offered under this catalog number
Note: This CCR7 cell line in the Chem-1 background replaces the CCR7-Chem-2 cell line previously offered under this catalog number
Background Information:
CCR7 is a GPCR that binds to CC chemokine ligands MIP-3beta (ELC/CCL19) and 6Ckine (CCL21) (Yoshida et al., 1997). These ligands are expressed in the secondary lymphoid organs, and binding to CCR7 expressed in naïve T cells, B cells and dendritic cells directs migration of these cells to sites of antigen presentation (Förster et al., 1999). Inhibition of CCR7/ligand interactions inhibits contact sensitivity, delayed type hypersensitivity, and graft vs host disease in experimental models (Förster et al., 1999; Sasaki et al. 2003). In addition, CCR7 expression by breast cancer, melanoma and other malignant cells are associated with lymph node metastasis (Muller et al., 2001; Payne and Cornelius, 2002). Chemicon's cloned human CCR7-expressing cell line is made in the Chem-2 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.
Application Notes:
Calcium flux assay, ligand binding assays
Species Reactivity:
Human
Usage Statement:
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.
Entrez Gene Summary:
The protein encoded by this gene is a member of the G protein-coupled receptor family. This receptor was identified as a gene induced by the Epstein-Barr virus (EBV), and is thought to be a mediator of EBV effects on B lymphocytes. This receptor is expressed in various lymphoid tissues and activates B and T lymphocytes. It has been shown to control the migration of memory T cells to inflamed tissues, as well as stimulate dendritic cell maturation. The chemokine (C-C motif) ligand 19 (CCL19/ECL) has been reported to be a specific ligand of this receptor.
UniProt Summary:
FUNCTION: SwissProt: P32248 # Receptor for the MIP-3-beta chemokine. Probable mediator of EBV effects on B-lymphocytes or of normal lymphocyte functions.
SIZE: 378 amino acids; 42874 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Expressed in various lymphoid tissues and activated B- and T-lymphocytes, strongly up-regulated in B-cells infected with Epstein-Barr virus and T-cells infected with herpesvirus 6 or 7.
SIMILARITY: SwissProt: P32248 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 378 amino acids; 42874 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Expressed in various lymphoid tissues and activated B- and T-lymphocytes, strongly up-regulated in B-cells infected with Epstein-Barr virus and T-cells infected with herpesvirus 6 or 7.
SIMILARITY: SwissProt: P32248 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Brand Family:
Chemicon
Protein Target:
CCR7
Presentation:
Cells are frozen at 2 x 106 cells/mL in RPMI/20% fetal bovine serum containing 10% DMSO. Cell line tests negative for mycoplasma.
Ligand Type:
peptide
Storage Conditions:
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.
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.
Target Sub-Family:
Chemokine
GPCR Class:
A
Promotional Text:
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Gene Symbol:
- CCR7
- CMKBR7
- EBI1
- CD197
- CDw197
- CCR-7
- BLR2
- EVI1
- CC-CKR-7
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiSCREEN™ CCR7 Calcium-Optimized FLIPR Cell Line
Materials Required but Not Delivered:
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)
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)
Quality Assurance:
EC50 for calcium mobilization by MIP-3β: 1.5 nM
EC50 for calcium mobilization by 6CKine: 5.9 nM
EC50 for calcium mobilization by 6CKine: 5.9 nM
Cell Type:
Chem-1
Cell Line Type:
GPCR Cell Lines
Host Cells:
Chem-1
Format:
Cell Line
UniProt Number:
Packaging:
2x106 cells/vial
Entrez Gene Number: