Patient-derived cultures of GBM cells, as well as U87MG cells, were grown in 96-well plates in media I and II at 37 ° C, 5% CO2 atmosphere, 100% humidity. Before calcium imaging began, the growing medium was replaced with extracellular buffer (140 mM NaCl, 2 mM CaCl2, 2.8 mM KCl, 4 mM MgCl2, 20 mM HEPES, 10 mM glucose, pH 7.4), and then each well was loaded with a cell-permeant 2,5 μM Fluo-4AM solution (ex/em = 494/506 nm; Thermo Fisher Scientific) for 40 min. The fluorescent dye solution was further removed, and cells were kept in an extracellular buffer for 1 h. Ca2+ dynamics were recorded using an Olympus IX71 epifluorescent microscope with an appropriate filter combination and a CAM-XM10 cooled CCD camera (Olympus, Tokyo, Japan). The cells were exposed to 10 μM of nAChR agonist acetylcholine iodide (Sigma-Aldrich), 1 μM of antagonists Azemiopsin, and [A10L]PnIA or RgIA (Syneuro, Moscow, Russia), and changes in the fluorescence of calcium indicator Fluo-4 were recorded for each cell independently. Videos were recorded and processed using CellA imaging software version 3.1 build 1274 (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Data analysis was performed using open-source ImageJ Fiji software (version 1.54f), where changes in fluorescent intensity per cell before and after the nAChR ligand exposure were calculated. The response of at least 5 cells was measured.
Fluo 4 am solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluo-4 AM solution is a fluorescent calcium indicator used to measure intracellular calcium levels in cells. It is a cell-permeant acetoxymethyl (AM) ester form of the fluorescent dye Fluo-4, which exhibits an increase in fluorescence upon binding to calcium ions.
Automatically generated - may contain errors
Lab products found in correlation
Ix81 spin disk confocal microscope, by Olympus (2 mentions)
Acetylcholine iodide, by Merck Group (1 mentions)
Ix71 epifluorescent microscope, by Olympus (1 mentions)
Cam xm10 cooled ccd camera, by Olympus (1 mentions)
Am251, by Bio-Techne (1 mentions)
Am630, by Bio-Techne (1 mentions)
96 well black clear bottom plate, by Greiner (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Softmax pro software, by Molecular Devices (1 mentions)
7 protocols using fluo 4 am solution
1
Calcium Imaging of nAChR Activation
2
Quantifying Calcium Dynamics in Cholangiocytes
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For calcium dye loading, cholangiocytes were incubated with 5 μM of Fluo4‐AM solution (Thermo Fisher Scientific) and placed on a rocker for 2 hours at 37°C. Excess dye was washed away with Hanks' balanced salt solution (HBSS) before flow experiments. The bile duct‐on‐a‐chip was connected to a syringe pump to apply shear flow with HBSS at 3.9 dyne/cm2. Time‐lapse images were acquired at 1‐second intervals for 3 minutes using an Olympus IX81 spin disk confocal microscope (Olympus, Tokyo, Japan).
3
Calcium Dye Imaging of Cholangiocytes
4
Cannabinoid Receptor Activation Assay
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In vitroCB1 and CB2 activity in OLC and HGF was determined by calculating changes in intracellular calcium concentrations ([Ca2+]i) after different stimuli. Firstly, cells were loaded with 2 μM of fluo-4 AM solution (Invitrogen, Thermo Fiseher Scientific) at 37 °C for 45 min in the dark. Next, cannabidiol 10 μM (CBD) (Biominerales Pharma, Colombia-Canada) was used for 1 and 10 min cell stimulation, either alone or with a 5-minute pretreatment with different antagonist concentrations (antagonist: agonist combinations, 10μM:10μM; 10μM:1nM; 1nM:1nM; 1nM:10μM), according to a previous viability experiment. A selective CB1 antagonist (AM251, Tocris, Bristol, UK), a selective CB2 inverse agonist (AM630, Tocris, Bristol, UK) and a TRPV1 antagonist (Capsazepine, CZP (Tocris, Bristol, UK) were used. The stock solution at a concentration of 10 mM was prepared in DMSO, and the serial dilutions used were prepared in culture medium.
Ionomycin is a lipophilic molecule that binds to calcium ions and carries them through membranes, so it was used as a positive control. Fluorescence quantitation of the cells was performed at a wavelength of 494/525 nm (excitation/emission) in a spectrofluorimeter (ClarioSTAR, BMG Labteeh). For the analysis of results, the data were normalized >with respect to unstimulated cells (F/FO) and plotted at each evaluation time (1 and 10 min).
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In vitroCB1 and CB2 activity in OLC and HGF was determined by calculating changes in intracellular calcium concentrations ([Ca2+]i) after different stimuli. Firstly, cells were loaded with 2 μM of fluo-4 AM solution (Invitrogen, Thermo Fiseher Scientific) at 37 °C for 45 min in the dark. Next, cannabidiol 10 μM (CBD) (Biominerales Pharma, Colombia-Canada) was used for 1 and 10 min cell stimulation, either alone or with a 5-minute pretreatment with different antagonist concentrations (antagonist: agonist combinations, 10μM:10μM; 10μM:1nM; 1nM:1nM; 1nM:10μM), according to a previous viability experiment. A selective CB1 antagonist (AM251, Tocris, Bristol, UK), a selective CB2 inverse agonist (AM630, Tocris, Bristol, UK) and a TRPV1 antagonist (Capsazepine, CZP (Tocris, Bristol, UK) were used. The stock solution at a concentration of 10 mM was prepared in DMSO, and the serial dilutions used were prepared in culture medium.
Ionomycin is a lipophilic molecule that binds to calcium ions and carries them through membranes, so it was used as a positive control. Fluorescence quantitation of the cells was performed at a wavelength of 494/525 nm (excitation/emission) in a spectrofluorimeter (ClarioSTAR, BMG Labteeh). For the analysis of results, the data were normalized >with respect to unstimulated cells (F/FO) and plotted at each evaluation time (1 and 10 min).
5
Intracellular Calcium Imaging using Fluo-4 AM
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Intracellular calcium was measured by imaging fluo-4 AM. Cells cultured on glass-bottomed tissue culture dishes (Corning incorporated, Corning, NY, United States) were rinsed by loading buffer (150 μM NaCl, 5 mM KCl, 1 μM MgCl, 10 μM glucose, and 20 μM HEPES, pH 7.4) twice and then bathed with fluo 4-AM solution (2.5 μM, Invitrogen Corp., Carlsbad, CA, United States), which is dissolved in loading buffer supplemented with 2% BSA and 0.01 % Pluronic F-127 (BASF) protected for light for 25 min at room temperature on a rolling plate. Cells were then washed twice using loading buffer and incubated in serum-free DMEM at 30°C for 20 min to allow the de-esterification.
6
Screening Ligand Activity in MOR-CHO Cells
7
Intracellular Calcium Imaging Protocol
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Cells are washed in HBSS post-treatment and Fluo4-AM solution (Invitrogen, 1 µM) was then loaded as mentioned by the manufacturer. After washing with calcium free HBSS, images are taken using fluorescence microscope (EVOS, Thermo). Signal intensity is measured by utilizing ImageJ.
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