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Human LPAR2/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0684
Cell Line Description: These cell lines are constructed in the Chem-1 host, which supports high levels of functional receptor expression on the cell surface. Chem-1 cells contain high endogenous levels of Galpha15, a promiscuous G protein, allowing most receptors to couple to the calcium signaling pathway. These LPA2 receptor-expressing cells were constructed by stable transfection of Chem-1 cells with LPA2 receptor and a promiscuous G protein to couple the receptor to the calcium signaling pathway. These stability-tested cells are ready for fluorescence-based assays for agonists, antagonists and modulators at the LPA2 receptor.
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. LPA2 is widely expressed, with particularly notable expression in helper T Cells. LPA2 upregulation has been demonstrated in multiple cancer cells, including colon and ovarian cancer. LPA2 couples to multiple signaling cascades through Gq, Gi/o and G12/13, mediating inhibition of adenyl cyclase, inositol phosphate turnover, calcium flux and cell migration.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of LPAR2.2. Protein expression: LPAR2 in this cell line has been validated by immunocytochemical staining.
Application: Calcium Flux 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: LPAR2 lysophosphatidic acid receptor 2 [ Homo sapiens ]
Official Symbol: LPAR2
Synonyms: LPAR2; lysophosphatidic acid receptor 2; EDG4, endothelial differentiation, lysophosphatidic acid G protein coupled receptor, 4; EDG 4; LPA2; LPA receptor 2; LPA receptor EDG4; G protein-coupled receptor; lysophosphatidic acid receptor EDG4; lysophosphatidic acid receptor Edg-4; endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 4; EDG4; EDG-4; LPA-2; FLJ93869;
Gene ID: 9170
mRNA Refseq: NM_004720
Protein Refseq: NP_004711
MIM: 605110
UniProt ID: Q9HBW0
Chromosome Location: 19p12
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; LPA receptor mediated events, organism-specific biosystem; Lysosphingolipid and LPA receptors, organism-specific biosystem;
Function: G-protein coupled receptor activity; LIM domain binding; PDZ domain binding; lipid binding; lysophosphatidic acid receptor activity; protein binding; receptor activity; signal transducer activity;

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