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Monkey CCR5/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0632
Cell Line Description: This 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 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 CCR5 and its ligands.
Background: CCR5 is the receptor for CC chemokines MIP-1alpha, MIP-1beta, and RANTES, and is preferentially expressed on Th1 lymphocytes. CCR5 is a coreceptor for macrophage-tropic HIV, and its ligands potently inhibit HIV replication in human leukocytes. In addition, HIV-infected patients with the nonfunctional CCR532 allele exhibit delayed onset of AIDS symptoms, and pharmacological antagonism of CCR5 inhibits HIV-1 infection. 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. 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.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of CCR5. 2. Protein expression: CCR5 in this cell line has been validated by immunocytochemical staining. 3. EC50 for calcium mobilization by MIP-1alpha: ~ 5.1 nM; EC50 for calcium mobilization by RANTES: ~ 2.0nM.
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: CCR5 chemokine (C-C motif) receptor 5 [ Macaca mulatta ]
Official Symbol: CCR5
Synonyms: C-C chemokine receptor type 5; CCR-5; CC-CKR-5; C-C CKR-5; CC chemokine receptor 5
Gene ID: 735311
mRNA Refseq: NM_001042773
Protein Refseq: NP_001036238
UniProt ID: P61813
Chromosome Location: 2
Pathway: Chemokine signaling pathway; Cytokine-cytokine receptor interaction; Endocytosis; Toxoplasmosis

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C-fusion    N-fusion   Non-tagged
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