Compound 89, also known as 2-((ethyl(4-fluorobenzyl)amino)methyl)-7,8-dimethylquinolin-4(1H)-one, was purchased from ChemDiv (San Diego, USA). Adenosine-5′-diphosphate monosodium salt, ATP disodium salt, UDP monosodium salt, and isoproterenol hydrochloride were purchased from Wako Pure Chemicals (Osaka, Japan). Uridine-5′-triphosphate was purchased from Sigma-Aldrich Japan (Tokyo, Japan). 2-Thio-UTP was purchased from Tocris Bioscience (Bristol, UK). Diadenosine 5′,5′″-P1,P4- tetraphosphate was purchased from Jena Bioscience (Jena, Germany).
2 thio utp
Manufactured by Bio-Techne
Sourced in United Kingdom
2-Thio-UTP is a nucleotide analog that serves as a precursor for in vitro transcription and labeling applications. It can be incorporated into RNA during enzymatic synthesis, enabling the production of modified RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Uridine 5 triphosphate, by Merck Group (1 mentions)
Sulprostone, by Cayman Chemical (1 mentions)
Oxytocin, by Bio-Techne (1 mentions)
Histamine, by Thermo Fisher Scientific (1 mentions)
Probenecid, by Merck Group (1 mentions)
Flexstation 3 multi mode microplate reader, by Molecular Devices (1 mentions)
Suramin, by Merck Group (1 mentions)
Lps strain 055 b5, by Merck Group (1 mentions)
Utpγs, by Bio-Techne (1 mentions)
Mwoi restriction enzyme, by New England Biolabs (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Medium 199, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Osmium tetroxide, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mrs 2693, by Bio-Techne (1 mentions)
Hepes, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
6 protocols using 2 thio utp
1
Compound 89 Synthesis and Characterization
2
Intracellular Calcium Assay for AsPC-1 Cells
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Intracellular
calcium concentration
of AsPC-1 cells was measured using the FLIPR-4 calcium assay reagent
(Cat # R8142, Molecular Devices, San Jose, CA). In brief, cells were
plated in black-walled clear-bottom 96-well plates overnight at ∼80%
confluency using media and conditions described inMethods . Culture media was then removed and cells were incubated
for 1 h at 37 °C, 5% CO2 in FLIPR-4 loading buffer,
consisting of FLIPR-4 reagent diluted (as per the manufacturer’s
instructions) in Hank’s balanced salt solution (HBSS) (with
calcium and magnesium) buffered with 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid and 0.2% bovine serum albumin,
with pH adjusted to 7.4. Loading buffer also contained probenecid
(2.50 mM, Sigma-Aldrich, Cat # P8761) to prevent leakage of the calcium
reagent from the cells. Calcium response was then measured via a FlexStation
3 Multi-Mode Microplate Reader (Molecular Devices). GPCR agonists
were added, and response in relative fluorescence units was measured
over 105 s for each well, yielding data for peak response and kinetics
of response. For calcium assays and wound-healing assays, we used
the following GPCR agonists: Neurotensin (Cat # 1909, Tocris, Minneapolis,
MN); 2-Thio-UTP (Cat # 3280, Tocris); histamine (Cat # AAJ6172703,
Fischer Scientific); oxytocin (Cat # 1910, Tocris); and sulprostone
(Cat # 14765, Cayman Chemical).
calcium concentration
of AsPC-1 cells was measured using the FLIPR-4 calcium assay reagent
(Cat # R8142, Molecular Devices, San Jose, CA). In brief, cells were
plated in black-walled clear-bottom 96-well plates overnight at ∼80%
confluency using media and conditions described in
for 1 h at 37 °C, 5% CO2 in FLIPR-4 loading buffer,
consisting of FLIPR-4 reagent diluted (as per the manufacturer’s
instructions) in Hank’s balanced salt solution (HBSS) (with
calcium and magnesium) buffered with 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid and 0.2% bovine serum albumin,
with pH adjusted to 7.4. Loading buffer also contained probenecid
(2.50 mM, Sigma-Aldrich, Cat # P8761) to prevent leakage of the calcium
reagent from the cells. Calcium response was then measured via a FlexStation
3 Multi-Mode Microplate Reader (Molecular Devices). GPCR agonists
were added, and response in relative fluorescence units was measured
over 105 s for each well, yielding data for peak response and kinetics
of response. For calcium assays and wound-healing assays, we used
the following GPCR agonists: Neurotensin (Cat # 1909, Tocris, Minneapolis,
MN); 2-Thio-UTP (Cat # 3280, Tocris); histamine (Cat # AAJ6172703,
Fischer Scientific); oxytocin (Cat # 1910, Tocris); and sulprostone
(Cat # 14765, Cayman Chemical).
3
Nucleotide and Cytokine Signaling Assay
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UTP, ATP, suramin, and LPS (strain 055:B5) were from Sigma-Aldrich (Ryde, Australia). 2-thio-UTP and UTPγS were from Tocris Biosciences (Bristol, UK). Interferon γ (IFN-γ) was from Roche Diagnostics. MwoI restriction enzyme was from NEB.
4
Nucleotide Reagents for Cell Culture
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UTP (uridine triphosphate), ATP (adenosine triphosphate), Dulbecco's modified Eagle's medium (DMEM), and 199 medium were purchased from Sigma-Aldrich (St. Louis, MO, USA); 2-thio-UTP and FMK-Z-YVAD were from Tocris (Bristol, UK).
5
Investigating Oxidative Stress Signaling Pathways
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Adenosine-50-triphosphate (ATP), adenosine diphosphate salt (ADP), uridine triphosphate salt (UTP), uridine diphosphate salt (UDP), MRS 2579, MRS 2693 and 2-Thio-UTP were from Tocris Bioscience (Bristol, UK). 40-6-diamidino-2-phenylindole (DAPI), hydrogenperoxide (H2O2), N-acetyl cysteine (NAC) and acridine orange, penicillin, streptomycin, HEPES, paraformaldehyde, glutaraldehyde, osmium tetroxide and bovine serum albumin (BSA) were purchased from Sigma Aldrich (St. Louis, USA). 20,70-dichlorofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE) were purchased from Calbiochem (USA). Lactate dehydrogenize commercial kit was purchased from DOLES (Brazil). Acetone and ethanol were from Merk (Darmstadt, Germany). Fetal bovine serum (FBS) was from Gibco/life technologies, USA. The anti-LAMP-1-PE monoclonal antibody (cat. no. 12-1071-82 mAb) conjugate was purchased from e-Bioscience (Brazil). The anti-SAG-1 polyclonal antibody (pAb) was kindly provided by Dr. J.C. Boothroyd (Stanford University, USA), and the goat anti-rabbit-Alexa Fluor 488 secondary antibody was from Life Technologies (USA).
6
Optimized Cell Culture Reagents
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Tissue culture reagents were purchased from Life Technologies (Paisley, UK); unless mentioned, all chemicals were purchased from Sigma-Aldrich (Poole, Dorset, UK). UTP and 2-thioUTP were purchased from Tocris Bioscience (Bristol, UK).
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