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Human C3AR1/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0613
Cell Line Description: This human C3aR-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant C3aR 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 C3aR and its ligand C3a.
Background: C3a, along with C4a and C5a, is a 77 amino acid anaphylotoxin generated by proteolytic cleavage during activation of the complement pathway. The anaphylotoxins strongly promote inflammation by recruiting leukocytes, particularly basophils, eosinophils, neutrophils and monocyte. The proinflammatory properties of C3a are mediated by interaction between the peptide and a 7-TM G-protein coupled receptor, C3aR. Genetic and pharmacological inhibition of C3a/C3aR interaction indicates an important role for C3aR in allergic asthma. C3a/C3aR also enhances the effect of SDF-1 in promoting retention of haematopoietic stem/progenitor cells within the bone marrow.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of C3AR1.2. Protein expression: C3AR1 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by C3a: ~ 8 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Anaphylatoxin
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: C3AR1 complement component 3a receptor 1 [ Homo sapiens ]
Official Symbol: C3AR1
Synonyms: C3AR1; complement component 3a receptor 1; C3a anaphylatoxin chemotactic receptor; AZ3B; C3AR; C3a-R; complement component 3 receptor 1; HNFAG09;
Gene ID: 719
mRNA Refseq: NM_004054
Protein Refseq: NP_004045
MIM: 605246
UniProt ID: Q16581
Chromosome Location: 12p13.31
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Complement and Coagulation Cascades, organism-specific biosystem; Complement and coagulation cascades, organism-specific biosystem; Complement and coagulation cascades, conserved biosystem; G alpha (i) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem;
Function: C3a anaphylatoxin receptor activity; G-protein coupled receptor activity; complement component C3a binding; complement component C3a receptor activity; phosphatidylinositol phospholipase C activity; receptor activity; signal transducer activity;

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