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Human PTGER3/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0739
Cell Line Description: This human EP3-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant EP3 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 EP3 and its ligands.
Background: Prostanoids are a series of arachidonic acid metabolites produced by the action of cyclooxygenase and subsequently 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, vasodilatation, immunosuppression of T cells, bone resorption 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. Further diversity is generated by alternative splicing; the human gene for EP3 generates 9 alternatively spliced mRNAs encoding 8 isoforms of EP3. These isoforms of EP3 vary in sequence at their C-termini, and differ in their ability to couple to Gs, Gq or Gi. EP3 is required for fever induced by pyrogens, a response long attributed to prostaglandins by the antipyretic action of aspirin and other COX inhibitors. In animal models of allergy, PGE2-mediated activation of EP3 inhibits inflammation to counteract the allergy-promoting activity of PGE2.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of PTGER3.2. Protein expression: PTGER3 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by PGE2: ~ 1 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Prostanoid
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: PTGER3 prostaglandin E receptor 3 (subtype EP3) [ Homo sapiens ]
Official Symbol: PTGER3
Synonyms: PTGER3; prostaglandin E receptor 3 (subtype EP3); prostaglandin E2 receptor EP3 subtype; EP3; prostanoid EP3 receptor; PGE receptor, EP3 subtype; PGE2 receptor EP3 subtype; prostaglandin receptor (PGE-2); prostaglandin E receotor EP3 subtype 3 isoform; EP3e; EP3-I; EP3-II; EP3-IV; PGE2-R; EP3-III; MGC27302; MGC141828; MGC141829;
Gene ID: 5733
mRNA Refseq: NM_001126044
Protein Refseq: NP_001119516
MIM: 176806
UniProt ID: P43115
Chromosome Location: 1p31.2
Pathway: Calcium signaling pathway, organism-specific biosystem; Calcium signaling pathway, conserved biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Eicosanoid ligand-binding receptors, organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem;
Function: G-protein coupled receptor activity; ligand-activated sequence-specific DNA binding RNA polymerase II transcription factor activity; prostaglandin E receptor activity; prostaglandin E receptor activity; 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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