HEK293FT cells were seeded at a density of 8 × 104 cells/well in a poly-d -lysine coated 48-well plate with DMEM containing 2% FBS. After overnight culture, cells were transfected with mock or pcDNA3.2-GPR55-V5 using PEI MAX and Opti-MEM for 24 h. GLUTag cells were seeded at a density of 8 × 104 cells/well in a poly-d -lysine coated 48-well plates with DMEM containing 10% FBS and incubated for 48 h. Cells were washed with standard extracellular buffer (Hepes-NaOH, pH7.5, 143.4 mM NaCl, 4.5 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, 5.5 mM glucose) containing 5 μM Fura-8AM (AAT Bioquest, Sunnyvale, CA, USA) for 15 min at 37 °C. After adaptation to room temperature (i.e., ~25 °C) for 15 min, the cells were washed three times and medium was replaced to fresh standard extracellular buffer containing 10 µM CID16020046 and incubated for 15 min, followed by stimulation of 10 μM (for HEK293FT cells) or 20 μM (for GLUTag cells) curcumin. Fluorescence intensity (Ex: 365 nm and Em: 530 nm) was determined with microplate reader (MTP-900, Corona Electric, Ibaraki, Japan) at 1-s intervals before and after stimulation with curcumin for 4 min. The integrated value of the intensity was calculated.
Fura 8am
Manufactured by AAT Bioquest
Sourced in United States
Fura-8AM is a fluorescent calcium indicator dye. It is used for monitoring intracellular calcium levels in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Arachidonic acid, by Merck Group (2 mentions)
Esculetin, by Merck Group (2 mentions)
Naproxen, by Merck Group (2 mentions)
3 ethoxy 4 methoxyphenol, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Mtp 900, by Corona Electric (1 mentions)
Cerulein, by Merck Group (1 mentions)
Collagenase p, by Merck Group (1 mentions)
Dantrolene sodium, by Merck Group (1 mentions)
U73122, by Merck Group (1 mentions)
2 aminoethoxydiphenyl borate 2 apb, by Merck Group (1 mentions)
Leukotriene b4, by Cayman Chemical (1 mentions)
Prostaglandin d2, by Merck Group (1 mentions)
Prostaglandin e2, by Merck Group (1 mentions)
Leukotriene b4 ltb4, by Cayman Chemical (1 mentions)
Axiovert 135 microscope, by Zeiss (1 mentions)
Glass bottom dishes, by Bioptechs (1 mentions)
5 protocols using fura 8am
1
Curcumin-Induced Calcium Signaling in Cell Lines
2
Calcium Signaling Assay Protocol
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Fura-8 AM was purchased from AAT Bioquest. CBA was from Tocris Bioscience (Bio-Techne Corporation). All other chemicals (including collagenase P, 9-ph, CPA, and cerulein) were obtained from Sigma–Aldrich (Merck).
3
Arachidonic Acid and Inflammatory Modulators
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Arachidonic acid (AA: 5,8,11,14-eicosatetraenoic acid), dexamethasone [DEX: (11β, 16α)-9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3], naproxen [NAP: (S)-(+)-2-(6-methoxy-2-naphthyl)propionic acid], esculetin (ESC: 6-hydroxy-7-methoxycoumarin), 3-ethoxy-4-methoxyphenol (EMP) were purchased from Sigma-Aldrich Korea (Seoul, Korea) and dissolved in dimethyl sulfoxide (DMSO). Leukotriene B4 (LTB4: 5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid) was purchased from Cayman Chemicals (Ann Arbor, MI, USA) and dissolved in DMSO. Prostaglandin E2 (PGE2: (5Z,11α,13E,15S)-11,15-dihydroxy-9-oxoprosta-5,13-dienoic acid), prostaglandin D2 (PGD2: 9α,15S-dihydroxy-11-oxoprosta-5Z,13E-dien-1-oic acid), 2-aminoethoxydiphenylborate (2-APB), dantrolene sodium (DAN: 1-[(5-(p-nitrophenyl) furfurylidene)amino] hydantoin sodium salt), and U-73122 (1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione) were purchased from Sigma-Aldrich Korea and dissolved in DMSO. Fura-8AM was purchased from AAT Bioquest (Sunnyvale, CA, USA) and dissolved in DMSO. Paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride hydrate) and vitamin C (L-ascorbic acid) were purchased from Sigma-Aldrich Korea and dissolved in distilled water. Phosphate-buffered saline (PBS) was prepared with 100 mM phosphoric acid and adjusted to pH 7.4.
4
Arachidonic Acid and Inflammation Modulators
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Arachidonic acid (AA: 5,8,11,14-eicosatetraenoic acid), dexamethasone [DEX (11β, 16α)-9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3], naproxen [NAP (S)-(+)-2-(6-methoxy-2-naphthyl)propionic acid], esculetin (ESC: 6-hydroxy-7-methoxycoumarin), 3-ethoxy-4-methoxyphenol (EMP) as a specific inhibitor to DSP1 [22 (link)], and PGE2 ((5Z,11α,13E,15S)-11,15-dihydroxy-9-oxoprosta-5,13-dienoic acid) were purchased from Sigma-Aldrich Korea (Seoul, Korea) and leukotriene B4 (LTB4: 5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). These chemicals were supplied in acetone, ethanol, or powder form. Chemicals in powder form were directly dissolved in dimethyl sulfoxide (DMSO) and stock concentrations (100 μg/µL or 105 ppm) were prepared. In case of chemicals in acetone or ethanol, they were dried with nitrogen gas under a fume hood and then prepared in stock concentrations (100 μg/µL or 105 ppm) with DMSO. Finally, a working solution (1,000 ppm) was prepared by a serial dilution using DMSO. Fura-8AM was purchased from AAT Bioquest (Sunnyvale, CA, USA) and dissolved in DMSO. Phosphate-buffered saline (PBS) was prepared with 100 mM phosphoric acid and adjusted to pH 7.4.
5
Fura-8 Calcium Imaging of Acinar Cells
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Acinar cell clumps were loaded with 2 μM Fura-8 AM (AAT Bioquest) Ca2+ sensitive dye for 30 min at room temperature. Acinar cells were plated on glass-bottom dishes (Bioptechs) and allowed to attach to the bottom. Cells were perfused with Tyrode's solution containing (in millimolar): 140 NaCl, 5 KCl, 2 MgCl2, 2.5 CaCl2, and 10 Hepes, and pH 7.38. Some experiments were performed with a Ca2+-free Tyrode's solution, which contained 0.5 EGTA, but no CaCl2. Ratiometric measurement of Fura-8 fluorescence was performed at room temperature using a Zeiss Axiovert 135 microscope equipped with a 40× Fluor (1.3 numerical aperture) objective. Fura-8 was excited at 360 and 405 nm at 1 Hz using an light-emitting diode light source (FuraLED; Cairn Research Ltd), and the emitted light was passed through a 520-nm longpass filter and collected using a Qimaging Retiga R3 charge-coupled device camera. The imaging hardware setup was controlled by Micromanager software (an open source software program) (61 (link), 62 ) through an interface. Fluorescence values were determined using ImageJ (National Institutes of Health) software with Fiji plugins. Fluorescence ratios of emissions elicited by excitations at 360 and 405 nm were calculated after background subtraction in single cells. Changes of ratios (ΔR) were determined for each cell, averaged, and presented as mean ± SEM.
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