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Figure 1. Calcium flux in CCR5-expressing Chem-1 cell line induced by MIP-1α and RANTES. CCR5-expressing Chem-1 cells and Wild-Type Chem-1 cells (Ch...
ChemiSCREEN™ Rhesus Macaque Recombinant CCR5 Chemokine Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Rhesus Macaque Recombinant CCR5 Chemokine Receptor Calcium-Optimized Stable Cell Line
Trade Name:
Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Full-length rhesus CCR5 cDNA encoding CCR5
Background Information:
CCR5 is the receptor for CC chemokines MIP-1α, MIP-1β, and RANTES (Raport et al., 1996), and is preferentially expressed on Th1 lymphocytes (Loetscher et al., 1998). CCR5 is a coreceptor for macrophage-tropic HIV, and its ligands potently inhibit HIV replication in human leukocytes (Cocchi et al., 1995). In addition, HIV-infected patients with the nonfunctional CCR532 allele exhibit delayed onset of AIDS symptoms (Samson et al., 1996), and pharmacological antagonism of CCR5 inhibits HIV-1 infection (Strizki et al., 2001). Preclinical testing of small molecule antagonists of CCR5 has been hampered by low affinity of the compounds to rodent and dog CCR5, but two such compounds, maraviroc and AD101, have been shown to have potent antagonist activity at rhesus macaque CCR5, which differs from human CCR5 by 8 amino acids (Napier et al., 2005; Billick et al., 2004). One antagonist of human CCR5, SCH-C, does not block HIV entry through rhesus macaque CCR5, and one amino acid difference is responsible for the functional difference (Billick et al., 2004). Millipore's cloned rhesus macaque CCR5-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant CCR5 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between CCR5 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:
This gene encodes a member of the beta chemokine receptor family, which is predicted to be a seven transmembrane protein similar to G protein-coupled receptors. This protein is expressed by T cells and macrophages, and is known to be an important co-receptor for macrophage-tropic virus, including HIV, to enter host cells. Defective alleles of this gene have been associated with the HIV infection resistance. The ligands of this receptor include monocyte chemoattractant protein 2 (MCP-2), macrophage inflammatory protein 1 alpha (MIP-1 alpha), macrophage inflammatory protein 1 beta (MIP-1 beta) and regulated on activation normal T expressed and secreted protein (RANTES). Expression of this gene was also detected in a promyeloblastic cell line, suggesting that this protein may play a role in granulocyte lineage proliferation and differentiation. This gene is located at the chemokine receptor gene cluster region. Two transcript variants encoding the same protein have been found for this gene.
UniProt Summary:
FUNCTION: SwissProt: P51681 # Receptor for a C-C type chemokine. Binds to MIP-1-alpha, MIP-1-beta and RANTES and subsequently transduces a signal by increasing the intracellular calcium ions level. May play a role in the control of granulocytic lineage proliferation or differentiation. Acts as a coreceptor (CD4 being the primary receptor) for HIV-1 R5 isolates.
SIZE: 352 amino acids; 40524 Da
SUBUNIT: Interacts with PRAF2. Interacts with HIV-1 surface protein gp120.
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Found in promyelocytic cells.
PTM: Sulfation contributes to the efficiency of HIV-1 entry. & O-glycosylated, but not N-glycosylated.
SIMILARITY: SwissProt: P51681 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 352 amino acids; 40524 Da
SUBUNIT: Interacts with PRAF2. Interacts with HIV-1 surface protein gp120.
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Found in promyelocytic cells.
PTM: Sulfation contributes to the efficiency of HIV-1 entry. & O-glycosylated, but not N-glycosylated.
SIMILARITY: SwissProt: P51681 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Rhesus Macaque
Brand Family:
Chemicon
Protein Target:
CCR5
Presentation:
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.
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 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.
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 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 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.
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.
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 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 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.
Target Sub-Family:
Chemokine
GPCR Class:
A
Promotional Text:
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Gene Symbol:
- CCR5
- CCCKR5
- CMKBR5
- chemr13
- CD195
- CKR5
- CHEMR13
- CC-CKR-5
- CKR-5
- CCR-5
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiSCREEN™ Rhesus Macaque Recombinant CCR5 Chemokine Receptor Calcium-Optimized Stable 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-1α: ~ 5.1 nM
EC50 for calcium mobilization by RANTES: ~ 2.0nM
EC50 for calcium mobilization by RANTES: ~ 2.0nM
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: