Myo 2 3h inositol

Manufactured by PerkinElmer

Sourced in United States

Myo-[2-3H]inositol is a radioactive labeled compound used for research purposes. It is a tritium-labeled form of the inositol molecule, which is a type of sugar alcohol. Myo-[2-3H]inositol is used as a tracer in various biochemical and cell biology studies to investigate the role of inositol and its derivatives in cellular processes.

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Lab products found in correlation

Anion exchange resin, by Bio-Rad (1 mentions) Prism 5, by GraphPad (1 mentions) Ag 1 x8 anion exchange resin, by Bio-Rad (1 mentions) Metafectene pro, by Biontex (1 mentions) Dulbecco s phosphate buffered saline dpbs, by Merck Group (1 mentions) A11010, by Thermo Fisher Scientific (1 mentions) A11003, by Thermo Fisher Scientific (1 mentions) A11008, by Thermo Fisher Scientific (1 mentions) Poly l lysine solution, by Merck Group (1 mentions) Trypsin edta, by Merck Group (1 mentions) Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions) Penicillin streptomycin, by GE Healthcare (1 mentions) Fetal bovine serum (fbs), by GE Healthcare (1 mentions) Totalchrom workstation, by PerkinElmer (1 mentions) Series 200 hplc system, by PerkinElmer (1 mentions) Partisphere sax column, by Cytiva (1 mentions) Opti mem 1, by Thermo Fisher Scientific (1 mentions) U46619, by Cayman Chemical (1 mentions) Sq29548, by Cayman Chemical (1 mentions) I bop, by Cayman Chemical (1 mentions)

Spelling variants of this product

[3H]myo-inositol (26 citations) 3H-inositol (5 citations) Myo-[2-3H(N)] inositol (3 citations)

9 protocols using myo 2 3h inositol

Ghrelin Receptor Functional Assay

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Briefly, 60,000 to 80,000 cells/well of COS7 cells, stably expressing the fusion protein hGhrR-eYFP, were seeded in 48-well plates in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS) and 0.4 mg/mL hygromycin B. The next day, the cells were labeled with [2-³H]-myo-inositol (2 µCi/mL, PerkinElmer). Stimulation with different concentrations of the compounds alone or with 10⁻⁸ M ghrelin together with different concentrations of the compounds (antagonist experiments) was performed in duplicates for 2 h [58 (link)]. Cell debris was removed and the supernatant purified on an anion-exchange resin (BioRad, Hercules, CA, USA). The obtained data were analyzed with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Dpm (decay per minute) values were normalized to ghrelin effects (100% = maximal efficacy, 0% = constitutive receptor activity). EC₅₀/IC₅₀ values were calculated from at least three independent experiments. E_max is defined as the difference between maximal and minimal effect in presence of the compound.

Moldovan R.P., Els-Heindl S., Worm D.J., Kniess T., Kluge M., Beck-Sickinger A.G., Deuther-Conrad W., Krügel U, & Brust P. (2017). Development of Fluorinated Non-Peptidic Ghrelin Receptor Ligands for Potential Use in Molecular Imaging. International Journal of Molecular Sciences, 18(4), 768.

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Measuring Intracellular cAMP and Inositol Phosphates in hTERT-HM Cells

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Basal and FSH-stimulated intracellular cAMP in hTERT-HM cells was measured as we described previously for other cells [24 (link)]. Briefly, cells were incubated with FSH at a saturating concentration with respect to cAMP accumulation (300 ng/ml, final) or vehicle only for 1 h at 37°C, at which point the intracellular concentration of cAMP was extracted and quantified by radioimmunoassay.
Basal and FSH-stimulated inositol phosphates in hTERT-HM cells were measured as previously described for other cells [4 (link), 25 (link)]. hTERT-HM cells were incubated in serum-free medium containing Ad-FSHR or Ad-β-gal as described above, after which the medium was removed and replaced with serum-containing growth medium supplemented with 4 μCi/ml [2-³H]myo-inositol (Perkin-Elmer). After incubation for 19 h, the cells were washed and incubated for 1 h at 37°C with 20 mM LiCl plus either vehicle or FSH at a saturating concentration with respect to accumulation of inositol phosphates (500 ng/ml, final). Inositol phosphates were extracted and quantified as described previously [4 (link), 25 (link)].

Stilley J.A., Guan R., Santillan D.A., Mitchell B.F., Lamping K.G, & Segaloff D.L. (2016). Differential Regulation of Human and Mouse Myometrial Contractile Activity by FSH as a Function of FSH Receptor Density. Biology of Reproduction, 95(2), 36.

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Ghrelin-Induced Inositol Phosphate Assay

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COS7 cells were transiently transfected with 4 μg receptor plasmid in 25 cm² culture flasks using 15 μl of MetafectenePro (Biontex, Munich, Germany) according to the manufacturer’s instructions. 24 h post-transfection, the cells were seeded into 48-well plates and grown to 90% confluency. The cells were labeled in DMEM supplied with 10% (v/v) FBS containing 2 μCi/ml myo-[2-³H]-inositol (Perkin Elmer, Waltham, USA) at 37°C in the presence of 5% CO₂ for 16-18 h. After aspirating the labeling solution, the cells were washed once with DMEM containing 10 mM LiCl and stimulated for 2 h with increasing ghrelin concentrations, ranging from 10⁻¹¹ M to 10⁻⁵ M in DMEM with 10 mM LiCl. Stimulation was stopped by aspiration of the medium followed by basic cell lysis with 0.1 M NaOH and subsequent neutralization with 0.13 M formic acid. After removal of the cell debris, radioactive inositol phosphate species were diluted and purified on an AG 1-X8 anion exchange resin (Bio-Rad, Hercules, USA) as described (Els et al., 2010 (link)). Radioactivity of the eluates was measured on a liquid scintillation counter. The experiments were repeated at least two times independently in technical triplicate.

Bender B.J., Vortmeier G., Ernicke S., Bosse M., Kaiser A., Els-Heindl S., Krug U., Beck-Sickinger A., Meiler J, & Huster D. (2019). Structural Model of Ghrelin Bound to its G Protein-Coupled Receptor. Structure (London, England : 1993), 27(3), 537-544.e4.

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Radiolabeled Inositol Derivatives Characterization

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myo-[2-³H]inositol (specific activity of 21 to 24 Ci mmol⁻¹; 777 to 888 GBq mmol⁻¹) was purchased from PerkinElmer Life Sciences. Inositol isomers and derivatives were obtained from Sigma Aldrich (St. Louis, MO) (allo-, d-chiro-, l-chiro-, epi-, muco-, and myo-inositol, phytic acid, and neo-inositol, and quebrachitol), Calbiochem (scyllo- and myo-inositol), or Industrial Research Ltd. (Lower Hutt, New Zealand) (l-chiro-inositol, d-ononitol, d-pinitol, and viburnitol). Monosaccharides, ionophores, and other inhibitors were purchased from Sigma Aldrich and were of the highest grade available.

Cushion M.T., Collins M.S., Sesterhenn T., Porollo A., Vadukoot A.K, & Merino E.J. (2016). Functional Characterization of Pneumocystis carinii Inositol Transporter 1. mBio, 7(6), e01851-16.

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Chemokinetic Signaling Receptor Assay

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Human chemokines CCL5 and CX3CL1 (PeproTech, Rocky Hill, NJ, USA). Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s Phosphate-buffered saline (D-PBS), trypsin-EDTA and Poly-L-lysine solution (Sigma-Aldrich, Saint Louis, MO, USA. Fetal bovine serum (FBS) and penicillin/streptomycin (PAA Laboratories GmbH, Cölbe, Germany). ¹²⁵I-Na and myo-[2-³H] inositol (1 mCi/ml) (Perkin Elmer Life Sciences, Waltham, MA, USA). Mouse anti-cytomegalovirus (immediate early antigen; MAB180R, Merck Millipore, Billerica, MA, USA). Polyclonal rabbit-anti-US28 antibodies were generated by Covance (Princeton, NJ, USA) as described previously [41 (link)]. Anti-Myc tag (clone 9B11, #2276, Cell Signaling Technology, Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated antibodies (1706515 and 1706516, Bio-Rad Laboratories, Hercules, CA, USA). Alexa Fluor-conjugated antibodies (A11001, A11003, A11008 and A11010, Thermo Fisher Scientific, Waltham, MA, USA).

Heukers R., Fan T.S., de Wit R.H., van Senten J.R., De Groof T.W., Bebelman M.P., Lagerweij T., Vieira J., de Munnik S.M., Smits-de Vries L., van Offenbeek J., Rahbar A., van Hoorick D., Söderberg-Naucler C., Würdinger T., Leurs R., Siderius M., Vischer H.F, & Smit M.J. (2018). The constitutive activity of the virally encoded chemokine receptor US28 accelerates glioblastoma growth. Oncogene, 37(30), 4110-4121.

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Inositol phosphate production measurement

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Inositol phosphate production was measured as described recently [24 (link)]. Briefly, HEK-293 cells labelled with 1 µCi/mL myo-2-[³H]-inositol (23.75 Ci/mmol; Perkin Elmer Life and Analytical Sciences, Rodgau-Jügesheim, Germany) were stimulated with 100 µM carbachol in HBSS containing LiCl for 20 min at 37 °C. The reaction was stopped by addition of 2 mL ice-cold methanol. The cells were scraped from the dishes, 1 mL H₂O and 2 mL chloroform were added, and the aqueous phase was transferred to Poly-Prep AG 1-X8 columns (Bio-Rad, Hercules, CA, USA). After washing with H₂O and 50 mM ammonium formate, inositol phosphates were eluted with 5 mL of 1 M ammonium formate and 0.1 M formic acid. The radioactivity was measured by liquid scintillation counting.

Blankenbach K.V., Claas R.F., Aster N.J., Spohner A.K., Trautmann S., Ferreirós N., Black J.L., Tesmer J.J., Offermanns S., Wieland T, & Meyer zu Heringdorf D. (2020). Dissecting Gq/11-Mediated Plasma Membrane Translocation of Sphingosine Kinase-1. Cells, 9(10), 2201.

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Quantification of Cellular Phosphoinositides

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Analysis of cellular phosphoinositide levels was performed as previously described (44 (link)) with the following modifications. Midlog cells (5 OD₆₀₀ equivalents) grown in yeast nitrogen base media were harvested, washed in inositol free media (IFM), incubated for 10 min in inositol-free media, and labeled with myo-[2-³H] inositol (PerkinElmer) for 30 min. Following this initial labeling, cultures were split and further incubated in IFM or IFM containing 1.2 M NaCl for 15 min. Cells were precipitated in 4.5% perchloric acid and lysed by vortexing with glass beads. Cell lysates were washed in 100 mM EDTA, phospholipids were deacylated, and further processed as previously described (44 (link)). Samples were dried and resuspended in H₂O. Identification and quantitation of ³H-labeled glycerolphosphoinositols was performed by HPLCy (HPLC) using a Series 200 HPLC system (PerkinElmer), a partisphere SAX column (HiChrom), an inflow 150TR radiomatic detector (PerkinElmer), TCNav software, and TotalChrom workstation (PerkinElmer). Data for individual phosphoinositide species data are shown as percentages of the total number of counts detected for normalization.

Malia P., Numrich J., Nishimura T., González Montoro A., Stefan C.J, & Ungermann C. (2018). Control of vacuole membrane homeostasis by a resident PI-3,5-kinase inhibitor. Proceedings of the National Academy of Sciences of the United States of America, 115(18), 4684-4689.

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Quantification of Inositol Phospholipids

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Vps34^fl/fl podocytes (35 mm dishes) were treated with Ad-Cre or empty Ad viruses as specified above. On day 4, cells were washed two times with inositol/glucose-free DMEM, containing 5% dialyzed FBS, 0.5% BSA, 5 μg/ml insulin and 5 μg/ml transferrin, 2 mM pyruvate, 25 mM HEPES, pH 7.4, and 100 units/ml penicillin and 100 μg/ml streptomycin for 24 h. Cells were then labeled with 25 μCi/ml myo-[2-³H]inositol (Perkin Elmer) in a fresh media, as detailed above, for 26 h. Cellular lipids were extracted, deacylated, and analyzed by HPLC (Waters 5215) on a 5-micron Partisphere SAX column (Whatman) as we described previously (5 (link),7 (link),14 (link),37 (link)). [³²P]GroPIns5P, [³²P]GroPIns3P, [³²P]GroPIns(4,5)P₂ and [³²P]GroPIns(3,5)P₂ prepared by enzymatic synthesis with [γ-³²P]-ATP were used as HPLC standards as described elsewhere (5 (link),7 (link),14 (link),37 (link)). Fractions were collected every 0.25 min and analyzed for [³H] and [³²P] radioactivity after addition of scintillation mixture. Data evaluation and documentation was performed by Microsoft EXCEL software. Individual peak radioactivity was quantified by area integration and presented as a percentage of the summed radioactivity from the [³H]-GroPIns3P, -4P, -5P, -(3,5)P₂, and -(4,5)P₂ peaks (“total radioactivity”).

Ikonomov O.C., Sbrissa D., Venkatareddy M., Tisdale E., Garg P, & Shisheva A. (2015). Class III PI 3-Kinase is the Main Source of PtdIns3P Substrate and Membrane Recruitment Signal for PIKfyve Constitutive Function in Podocyte Endomembrane Homeostasis. Biochimica et biophysica acta, 1853(5), 1240-1250.

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Cell Culture and Molecular Biology Reagents

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Cell-culture media and supplements, animal serum, antibiotics, Lipofectamine® 2000, Opti-MEM I and molecular biology reagents were purchased from Invitrogen (Carlsbad, CA).
Inositol-free Dulbecco's modified Eagle's medium (DMEM) was from ICN Pharmaceuticals Inc. (Costa Mesa, CA). Ultima Gold™ was from PerkinElmer Life and Analytical Sciences (Boston, MA), as were [5,6-3 H]SQ29,548 and myo-[2-3 H]inositol. U46619, I-BOP, 8-isoPGF 2α (8-iso Prostaglandin F 2α ), 8-isoPGE 2 (8-iso Prostaglandin E 2 ), PTA2, SQ29,548 and Ramatroban were from Cayman Chemical (Ann Arbor, MI). The Renilla Luciferase substrate for BRET 2 , coelenterazine 400A was from Biotium (Hayward, CA). Anion exchange resin AG 1X-8 (formate form, 200-400 mesh) and Lowry dye-binding protein reagents were from Bio-Rad (Hercules, CA). All other reagents of the highest purity were available from Sigma-Aldrich (St. Louis, MO).

Capra V., Mauri M., Guzzi F., Busnelli M., Accomazzo M.R., Gaussem P., Nisar S.P., Mundell S.J., Parenti M, & Rovati G.E. (2017). Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo. Biochemical pharmacology, 124.

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