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Human LPAR1/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0682
Cell Line Description: This human LPA1-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant LPA1 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 LPA1 and its ligands.
Background: Lysophosphatidic acid (LPA) is a lysophospholipid produced by activated platelets that inhibits adenylate cyclase and stimulates DNA synthesis, changes in cell morphology, and increases in intracellular calcium in a variety of cultured mammalian cells. A family of three GPCRs, LPA1, LPA2 and LPA3, mediates the biological effects of LPA. LPA1 is widely expressed, with particularly notable expression in the ventricular zone of the embryonic cerebral cortex. Mice lacking LPA1 have changes in brain 5-HT and amino acids, and craniofacial abnormalities.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of LPAR1.2. Protein expression: LPAR1 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by Oleoyl-L-alpha-lysophosphatidic acid: ~ 180 nM; IC50 for Ki16425: ~65nM; Signal/Noise at ligand Emax: 224; Z at ligand EC80: 0.83.
Application: Calcium flux assay, ligand binding assays
Sub-type: Lysophospholipid
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: LPAR1 lysophosphatidic acid receptor 1 [ Homo sapiens ]
Official Symbol: LPAR1
Synonyms: LPAR1; lysophosphatidic acid receptor 1; EDG2, endothelial differentiation, lysophosphatidic acid G protein coupled receptor, 2; edg 2; Gpcr26; GPR26; LPA1; Mrec1.3; rec.1.3; vzg 1; LPA-1; LPA receptor 1; ventricular zone gene 1; lysophosphatidic acid receptor Edg-2; endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2; EDG2; VZG1; edg-2; vzg-1;
Gene ID: 1902
mRNA Refseq: NM_001401
Protein Refseq: NP_001392
MIM: 602282
UniProt ID: Q92633
Chromosome Location: 9q
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; G alpha (q) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; Gap junction, organism-specific biosystem; Gap junction, conserved biosystem;
Function: G-protein alpha-subunit binding; G-protein coupled receptor activity; PDZ domain binding; lysophosphatidic acid receptor activity; phospholipid binding; protein binding; receptor activity; signal transducer activity;

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