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Human PTGIR/Clytin Stable Cell Line-HEK293

Cat.No.: CSC-RG0745
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. This human IP1 receptor-expressing cells are made by stable transfection of HEK293 cells with clytin and IP1 Receptor. These stability tested cells are ready for luminescent analysis of agonists, antagonists and modulators at the IP1 receptor.
Background: Prostacyclin (PGI2) is released by vascular endothelial cells and serves as a potent vasodilator, inhibitor of platelet aggregation, and moderator of vascular smooth muscle cell proliferation–migration–differentiation. The function of prostacyclin is mediated via a seven transmembrane GPCR, IP1, which is known to couple to Gs and Gq signaling pathways. Mice lacking the IP1 receptor have shown increased susceptibility to thrombosis, enhanced injury-induced vascular proliferation and platelet activation, as well as reperfusion injury. The recent world-wide withdrawal of selective COX-2 inhibitors, rofecoxib(Vioxx) and valdecoxib(Bextra) is also due to their discriminating suppression of COX-2-derived prostacyclin and IP1-mediated cardioprotective effects, leading to increased risk of cardiovascular events.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of PTGIR.2. Protein expression: PTGIR in this cell line has been validated by immunocytochemical staining.
Application: Calcium Flux Assays; 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: PTGIR prostaglandin I2 (prostacyclin) receptor (IP) [ Homo sapiens ]
Official Symbol: PTGIR
Synonyms: PTGIR; prostaglandin I2 (prostacyclin) receptor (IP); prostacyclin receptor; IP; PGI receptor; PGI2 receptor; prostanoid IP receptor; prostaglandin I2 receptor; PRIPR; MGC102830;
Gene ID: 5739
mRNA Refseq: NM_000960
Protein Refseq: NP_000951
MIM: 600022
UniProt ID: P43119
Chromosome Location: 19q13.3
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; Hemostasis, organism-specific biosystem;
Function: G-protein coupled receptor activity; guanyl-nucleotide exchange factor 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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