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Human CCR5/Gi Stable Cell Line-CHO

Cat.No.: CSC-RG0492
Cell Line Description: These GPCR stable cell lines are non-force coupled cell lines that are validated for functional cAMP response and are fully characterized for pharmacology & specificity. Each clonal cell line is engineered to express the full length GPCR protein. These GPCR stable cell lines are used with cAMP detection kit for measuring the activation of the target (GPCR).
Background: CCR5 (chemokine (C-C motif) receptor 5) is a receptor for C-C type chemokines MIP-1-alpha, MIP-1-? and CCL5. CCR5 plays a role in the control of granulocytic lineage proliferation or differentiation. It also acts as co-receptor with CD4 for primary non-syncytium-inducing strains (macrophage-tropic) of HIV-1 virus.
Growth Properties: Adherent
Morphology: Epithelial-like
Host Cell: CHO-K1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of human CCR5.2. Protein expression: CCR5 in this cell line has been validated by immunocytochemical staining.
Application: cAMP assays
Sub-type: Chemokine
Propagation: Complete growth medium: F12 Nutrient Mixure (HAM) + 10% FBS + 1× penicillin + 1× streptomycin + 1× L-glutamine + 800μg/ml geneticin. Atmosphere: air, 95%; carbon dioxide (CO2), 5%. Temperature: 37 °C
Starting Cells From Frozen Cell Stock: 1. Thaw frozen cells very briefly in a 37 °C water bath under sterile conditions until just before ice completely melts (30 seconds to 1 minute). Caution: Longer incubation may result in cell death.2. Remove DMSO from the media by carefully transferring thawed cells to a sterile 15 mL tube, filling tube with complete media without antibiotics pre-warmed to 37 °C, and centrifuge at 300g for 4 minutes to pellet cells.3. Resuspend cell pellet in 5 mL of pre-warmed complete media without antibiotics, transfer to a T25 flask, and grow for 24 hours. Cell recovery is greatly improved when antibiotics are omitted for the first 24 hours.4. After 24 hours, exchange with 5 mL of media containing antibiotics. Antibiotic selection must be applied after the first 24 hours or the expression of GPCR could be lost. 5. Once the cells become >70% confluent in the T25 flask, trypsinize (using a 0.05% trypsin solution) and resuspend with 5 mL of complete media. Transfer the entire cell suspension to a T75 flask containing 5 mL ofcomplete media (containing antibiotics) for continued growth.
Subculturing: 1. Remove and discard culture medium.2. Wash cells with PBS (pH=7.4) to remove all traces of serum that contains trypsin inhibitor.3. Add 2.0 ml of 0.05% (w/v) Trypsin-EDTA (GIBCO, Cat No. 25300) solution to 10 cm dish and observe the cells under an inverted microscope until cell layer is dispersed (usually within 3 to 5 minutes). Note: To avoid clumping, do not agitate the cells by hitting or shaking the dish while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37 °C to facilitate dispersal.4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting, centrifuge the cells at 200g for 5min, and discard the medium.5. Resuspend the cells in culture medium and add appropriate aliquots of the cell suspension to new culture vessels.6. Incubate cultures at 37°C.Subcultivation Ratio: 1:3. Medium Renewal: Every 2 to 3 days.
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Complete growth medium 95%; DMSO, 5%
Storage: Store at -80 °C for less than 2 weeks. Store in vapor phase of liquid nitrogen for >2 weeks.
Preservation: 1. Detach cells from culture dish according to the Sub-Culture Procedure.2. Resuspend cells at a density of 5 x 10^6 cells/mL in freeze medium.Note: A T-75 culture flask typically yields enough cells for preparing two frozen vials.3. Aliquot 1 mL cells into cryogenic vials.4. Place vials in a freezing container and store at –80 °C overnight.5. Transfer vials to liquid nitrogen for long term storage. If properly stored, cells should remain stable for years.
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 (gene/pseudogene) [ Homo sapiens ]
Official Symbol: CCR5
Synonyms: CCR5; chemokine (C-C motif) receptor 5 (gene/pseudogene); chemokine (C C motif) receptor 5 , CMKBR5; C-C chemokine receptor type 5; CC CKR 5; CD195; CKR 5; CKR5; IDDM22; chemr13; HIV-1 fusion coreceptor; chemokine receptor CCR5; C-C motif chemokine receptor 5 A159A; CCR-5; CKR-5; CCCKR5; CMKBR5; CC-CKR-5; FLJ78003;
Gene ID: 1234
mRNA Refseq: NM_000579
Protein Refseq: NP_000570
MIM: 601373
UniProt ID: P51681
Chromosome Location: 3p21
Pathway: Binding and entry of HIV virion, organism-specific biosystem; 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;
Function: C-C chemokine binding; C-C chemokine receptor activity; C-C chemokine receptor activity; G-protein coupled receptor activity; actin binding; chemokine (C-C motif) ligand 5 binding; chemokine receptor activity; coreceptor activity; phosphatidylinositol phospholipase C activity; protein binding; receptor activity; signal transducer activity;

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Optional requirements on this protein    +Expand
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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