Cortisol, EGTA (ethylene glycol bis (β-amino ethyl ether)-N,N,N′,N′-tetraacetic acid), trypan blue, cholesterol, methyl-beta-cyclodextrin (MβCD), latrunculin B, and Cpd5J-4 were purchased from Sigma, St. Louis, MO, USA. Pluoronic®-F 127 (20% solution in DMSO), Fura 2-AM, and Laurdan were purchased from Thermofisher, Waltham, MA, USA. Cortisol-conjugated BSA was purchased from EastCoast Bio, North Berwick, ME, USA. All other chemicals were of analytical grade and were purchased from local suppliers.
Pluoronic f127
Manufactured by Thermo Fisher Scientific
Sourced in United States
Pluronic F127 is a non-ionic surfactant composed of polyethylene oxide and polypropylene oxide. It is commonly used in various biomedical and pharmaceutical applications as a solubilizing agent, emulsifier, and dispersant.
Automatically generated - may contain errors
Lab products found in correlation
Popo3 iodide, by Thermo Fisher Scientific (3 mentions)
Cal 520, by Abcam (3 mentions)
Hanks balanced salt solution (hbss), by Merck Group (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Laurdan, by Thermo Fisher Scientific (1 mentions)
Cortisol, by Merck Group (1 mentions)
Latrunculin b, by Merck Group (1 mentions)
Methyl β cyclodextrin, by Merck Group (1 mentions)
Epifluorescent microscope, by Zeiss (1 mentions)
8 protocols using pluoronic f127
1
Cortisol Signaling Pathway Assay
2
Calcium Imaging of Cardiac Microtissues
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6 aCM MTs and vCM MTs per experiment were allowed to sediment by gravity and loaded with 8 µm Rhod-2 AM fluorescent calcium indicator (Thermo Fisher) and 0.02% Pluoronic F-127 (Thermo Fisher) in Tyrode's solution containing 140 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 10 mm HEPES and 10 mm glucose (pH 7.4) for 20 min at room temperature. Cardiac microtissues were subsequently washed with Tyrode's solution before being acquired on a Zeiss Epi fluorescent microscope. Post-acquisition analysis was carried out using Caltrack, a MatLab plugin for intracellular Ca2+ analysis (69 (link)).
3
Intracellular Ca2+ Imaging in RASF
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In black 96-well plates, RASF were incubated with 4 µM of calcium dye Cal-520 (ab171868, abcam, Cambridge, UK) in Hanks buffered salt solution (1.66 mM Ca2+) (HBSS, sigma, # 55037C) or PBS (no Ca2+) with 0.02% Pluoronic F127 (Thermo fisher scientific, Waltham, MA, USA, # P6866) for 60 min at 37 °C followed by 30 min at room temperature. After washing, HBSS or PBS containing 1 µM PoPo3 iodide (Thermo fisher scientific, # P3584) and respective antagonists/ligands/inhibitors were added for 30 min at room temperature. After that, CBG was added and the intracellular Ca2+ concentration as well as PoPo3 uptake were evaluated with a TECAN multimode reader over 90 min.
4
Thioesterase Activity Assay for APT
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The thioesterase activity assay was modified from Kemp et al. 35 (link). Recombinant APT was preincubated for 10 min at room temperature with either DMSO or inhibitor in DMSO, where applicable. The reaction was initiated by the addition of 4-methylumbelliferyl palmitate (Santa Cruz Biotechnology sc-214256, PubChem CID 87248) and Pluoronic® F-127 (ThermoFisher P3000MP, PubChem CID24751). Enzyme activity was monitored by the appearance of methylumbelliferone over time (Ex 360 nm, Em 449 nm). The assay was optimized to use 0.4 µM of enzyme, 0.1 mM of substrate, and 0.1% detergent in HBuffer for a 96-well plate format. The following compounds were used as inhibitors: ML348 (TOCRIS 5345, PubChem CID 3238952), ML349 (TOCRIS 5344, PubChem CID 16193817), and 2-Bromopalmitate (Fisher Scientific AC218610500, PubChem CID 82145).
5
Calcium Dynamics and Dye Uptake in RASF
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In black 96-well plates, RASF were incubated with 4 µM of calcium dye Cal-520 (ab171868, Abcam, Cambridge, UK) in PBS with 0.02% Pluoronic F127 (Thermo Fisher scientific, Waltham, MA, USA, # P6866) for 60 min at 37 °C, followed by 30 min at room temperature. After washing, HBSS or PBS containing 1 µM PoPo3 iodide (Thermo Fisher scientific, # P3584) and respective antagonists/ligands/inhibitors were added for 30 min at room temperature. After that, THC was added and the intracellular Ca2+ concentration as well as PoPo3 uptake were evaluated with a TECAN multimode reader over 90 min.
6
Intracellular Calcium Dynamics Monitoring
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In black 96-well plates, RASF were incubated with 4 µM of calcium dye Cal-520 (ab171868, abcam, Cambridge, UK) in Hanks buffered salt solution (1 mM Ca2+; HBSS, sigma, # 55037 C) or PBS (no Ca2+) with 0.02% Pluoronic F127 (Thermo fisher scientific, Waltham, USA, # P6866) for 60 min at 37 °C followed by 30 min at room temperature. After washing, HBSS or PBS containing 1 µM PoPo3 iodide (Thermo fisher scientific, # P3584) and respective antagonists/ligands/inhibitors were added for 30 min at room temperature. After that, CBD was added and the intracellular Ca2+ concentration as well as PoPo3 uptake were evaluated with a TECAN multimode reader over 90 min.
7
Thioesterase Activity Assay for APT
8
Thioesterase Activity Assay for APT
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The thioesterase activity assay was modified from Kemp et al. (Kemp et al, 2013) .
Recombinant APT was preincubated for 10 min at room temperature with either DMSO or inhibitor in DMSO, where applicable. The reaction was initiated by the addition of 4-methylumbelliferyl palmitate (Santa Cruz Biotechnology sc-214256, PubChem CID 87248) and Pluoronic® F-127 (ThermoFisher P3000MP, PubChem CID24751). Enzyme activity was monitored by the appearance of methylumbelliferone over time (Ex 360 nm, Em 449 nm). The assay was optimized to use 0.4 µM of enzyme, 0.1 mM of substrate, and 0.1% detergent in HBuffer for a 96-well plate format.
The following compounds were used as inhibitors: ML348 (TOCRIS 5345, PubChem CID 3238952), ML349 (TOCRIS 5344, PubChem CID 16193817), and 2bromopalmitate (Fisher Scientific AC218610500, PubChem CID 82145).
Recombinant APT was preincubated for 10 min at room temperature with either DMSO or inhibitor in DMSO, where applicable. The reaction was initiated by the addition of 4-methylumbelliferyl palmitate (Santa Cruz Biotechnology sc-214256, PubChem CID 87248) and Pluoronic® F-127 (ThermoFisher P3000MP, PubChem CID24751). Enzyme activity was monitored by the appearance of methylumbelliferone over time (Ex 360 nm, Em 449 nm). The assay was optimized to use 0.4 µM of enzyme, 0.1 mM of substrate, and 0.1% detergent in HBuffer for a 96-well plate format.
The following compounds were used as inhibitors: ML348 (TOCRIS 5345, PubChem CID 3238952), ML349 (TOCRIS 5344, PubChem CID 16193817), and 2bromopalmitate (Fisher Scientific AC218610500, PubChem CID 82145).
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