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Human PTGER4/Clytin Stable Cell Line-HEK293

Cat.No.: CSC-RG0742
Cell Line Description: These cells express a novel variant of clytin, a calcium-activated photoprotein, to enable sensitive luminescent detection of ligand-induced calcium flux. The clytin contains a mutation that increases its affinity for calcium to a level that permits detection of cytosolic calcium in many cells with greater sensitivity than other mitochondrially expressed photoproteins. Luminescent calcium assays offer several advantages over fluorescent calcium assays including increased sensitivity and lack of interference from fluorescent compounds. These EP4 receptor-expressing cells are made by stable transfection of HEK293 cells with clytin, EP4 receptor and a promiscuous G protein to couple the receptor to the calcium signaling pathway. These stability tested cells are ready for luminescent analysis of agonists, antagonists and modulators at the EP4 receptor.
Background: Prostanoids are a series of arachidonic acid metabolites produced by the action of cyclooxygenase and further modified by isomerases and synthases. Cells rapidly secrete prostanoids after synthesis, whereupon the prostanoids bind to a family of 8 GPCRs to exert their biological effects. The prostaglandin PGE2 causes pain, vasodilation, immunosuppression of T cells, bone remodeling and promotion of carcinogenesis. Four related GPCRs: EP1, EP2, EP3 and EP4, each bind to PGE2, but the different G protein coupling status of each receptor leads to distinct biological effects. EP4 couples primarily to Gs to increase intracellular cAMP levels. During neonatal development, EP4 participates in closure of the ductus arteriosus, a process required for switching circulation from the placenta to the lungs. In addition, EP4 mediates PGE2-induced bone formation by promoting osteoblastogenesis, and selective EP4 agonists are being evaluated as potential treatments for osteoporosis.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of PTGER4.2. Protein expression: PTGER4 in this cell line has been validated by immunocytochemical staining.
Application: Calcium Flux and cAMP Assays
Sub-type: Prostanoid
Propagation: Complete growth medium: DMEM/F12 with 2.5 mM glutamine, 10% heat-inactivated FBS, 1x Nonessential amino acids, 1x Pen-Strep, 250μg/mL Genetecin/G-418Plating medium: DMEM/F12 with 2.5 mM glutamine, 10% heat-inactivated FBS, 1x Nonessential amino acids, 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. 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 Accutase at 1 mL/T75 and keep at room temperature until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize Accutase by addition of 4 mL Growth Media per 1 mL Accutase. 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: 90% heat-inactivated FBS + 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: PTGER4 prostaglandin E receptor 4 (subtype EP4) [ Homo sapiens ]
Official Symbol: PTGER4
Synonyms: PTGER4; prostaglandin E receptor 4 (subtype EP4); prostaglandin E2 receptor EP4 subtype; EP4; prostanoid EP4 receptor; PGE receptor EP4 subtype; PGE receptor, EP4 subtype; PGE2 receptor EP4 subtype; EP4R; MGC126583;
Gene ID: 5734
mRNA Refseq: NM_000958
Protein Refseq: NP_000949
MIM: 601586
UniProt ID: P35408
Chromosome Location: 5p13.1
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Eicosanoid ligand-binding receptors, organism-specific biosystem; G alpha (s) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class A Rhodopsin-like, organism-specific biosystem; Neuroactive ligand-receptor interaction, organism-specific biosystem;
Function: G-protein coupled receptor activity; prostaglandin E receptor 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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