To study the effect of oxalate on changes in intracellular Ca2+ ([Ca2+]i) levels, cells were treated with 0.5 mM of oxalate in combination with TRPV1 blocker SB or Ca2+ chelator BAPTA for 24 h, as described previously [54 (link)]. After being washed with 0.01 M PBS (pH 7.4) 3 times, treated cells were incubated for 30 min with 4 μM of cell-permeant fluorescent dye Fluo-3 acetoxymethyl ester (Molecular Probes, Eugene, OR, USA) with 0.04% dimethyl sulfoxide (DMSO) and 0.02% pluronic acid at 37 °C. Cells were then imaged by an inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a fluorescence image analytic system (Diagnostic Instruments, Sterling Heights, MI, USA) with the filter under excitation of 488 nm and emission of 522 nm to detect fluorescence intensity, as described previously [55 (link)].
Fluo 3 acetoxymethyl ester
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluo-3-acetoxymethyl ester is a fluorescent calcium indicator used for measuring intracellular calcium levels in living cells. It is a cell-permeable dye that can be loaded into cells and used to detect and monitor changes in calcium concentration over time.
Automatically generated - may contain errors
Lab products found in correlation
Pluronic f 127, by Thermo Fisher Scientific (2 mentions)
Pluronic f127, by BASF (2 mentions)
Inverted microscope, by Leica (1 mentions)
Carfilzomib, by Selleck Chemicals (1 mentions)
Chop gadd153, by Cell Signaling Technology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Ethidium homodimer 1 ethd 1, by Thermo Fisher Scientific (1 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Protein disulfide isomerase pdi, by Enzo Life Sciences (1 mentions)
Nutlin 3, by Bio-Techne (1 mentions)
Rhod 2 am, by Thermo Fisher Scientific (1 mentions)
Bapta am, by Merck Group (1 mentions)
Bortezomib, by Selleck Chemicals (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Rsc 200 rapid solution changer, by Bio-Logic (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
7 protocols using fluo 3 acetoxymethyl ester
1
Oxalate-induced Intracellular Calcium Changes
2
Mitochondrial Calcium Regulation in Cell Stress
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The chemicals used in this study were cycloheximide (CHX), MG132, 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) (Sigma-Aldrich, St Louis, MO, USA); Nutlin-3 (TOCRIS, Avonmouth, Bristol, UK); Rhod-2-acetoxymethyl ester (Rhod-2-AM), Fluo-3-acetoxymethyl ester (Fluo-3-AM), MitoTracker-Red (MTR), MitoTracker-Green (MTG), calcein-acetoxymethyl ester (calcein-AM), ethidium homodimer-1 (EthD-1), and 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probe, Eugene, OR, USA); Ru360 (Calbiochem, Darmstadt, Germany); bortezomib, and carfilzomib (Selleckchem, Houston, TX, USA). The antibodies used in this study were anti-β-actin, ubiquitin, ATF4, mitochondrial Ca2+ uniporter (MCU), HDM2 and p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); CHOP/GADD153, GRP78, and mtHsp70 (Cell Signaling Technology, Danvers, MA, USA); cytochrome oxidase subunit II (COX II) (Invitrogen, Grand Island, NY, USA); protein disulfide isomerase (PDI) (Enzo Life Sciences, Farmingdale, NY, USA); rabbit IgG HRP, mouse IgG HRP and goat IgG HRP (Molecular Probe).
3
Intracellular Calcium Measurement in MCF-7 Cells
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The analysis of [Ca 2 þ ] i was performed as previously reported (Formigli et al., 2002) . Briefly, to reveal the resting intracellular calcium concentration in MCF-7 cells in the absence or presence of melatonin (100 μM), $ 2 Â 10 4 cells were plated on glass coverslips and incubated at 37 1C for 10 min in DMEM with Fluo 3-acetoxymethyl ester (Molecular Probes) as fluorescent Ca 2 þ indicator at a final concentration of 10 μM and 0.1% anhydrous dimethyl sulfoxide and Pluronic F-127 (0.01% wt/vol) as dispersing agent (Molecular Probes). After being washed, the cells were placed in open slide flow-loading chambers mounted on the stage of the confocal laser scanning microscope. Optical sections (1024 Â 1024 pixels) at intervals of 0.8 mm were obtained. A variable number of cells ranging from 15 to 30 were analysed for each cell preparation. Multiple regions of interest (ROIs) of 25 μm 2 were selected within the cells to monitor Ca 2 þ signals, and outside the cells as baseline. Fluorescence signals were expressed as fractional changes above the resting baseline, ΔF/F, where F was the averaged baseline fluorescence and ΔF represented the fluorescence changes from the baseline.
4
Fluorescence Imaging of Neuronal Activity
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Primary TG cells kept in culture for 2 days were rinsed with basic solution (BS) containing (in millimolar) 152 NaCl, 2.5 KCl, 10 HEPES, 10 glucose, 2 CaCl2, 1 MgCl2 (pH adjusted to 7.4) followed by loading with 2 µM fluo-3 acetoxymethyl ester (Invitrogen, USA) at 37°C for 45 min in BS. After 20 min postincubation, dishes were transferred to TILL Photonics imaging system (TILL Photonics GmbH, Munich, Germany) and were constantly perfused (at 1.2 ml/min) with BS. The setup was equipped by fast perfusion system (Rapid Solution Changer RSC-200, BioLogic Science Instruments, Grenoble, France), which allowed rapid (exchange time ~30 ms) application of various compounds. Cells were viewed via Olympus IX-70 (Tokyo, Japan) microscope with a specific filter of 488 nm wavelength using 10× objective. Images were collected using CCD camera (SensiCam, PCO imaging, Kelheim, Germany) at sampling frequency set to 2 fps. Cells were further characterized by their responsiveness to a brief application of high potassium (50 mM KCl with compensated osmolarity) as a marker for neurons.
5
Calcium Transient Measurement in Myocytes
6
Calcium Transient Measurement in Myocytes
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Calcium measurements were carried out as previously described7 with the following modifications. Myocytes were loaded with 1μM Fluo-3-acetoxymethyl ester (Invitrogen) and 15% Pluronic F127 (a poloxamer made by BASF, Florham Park, NJ, USA) for 15 minutes. Cells were scanned using a 488- nm argon ion laser in confocal line-scan mode at 0.909 ms/line. Cells were electrically paced at 0.5 Hz at room temperature for 30 s to achieve steady state; five steady-state transients of each myocyte were averaged, pooled in groups and analyzed for calcium transient properties. The measured fluorescence (F) throughout the transient was normalized to the resting fluorescence prior to stimulation (F0) to normalize for heterogeneity in dye loading.
7
Calcium Imaging for Intracellular Ca2+ Measurement
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Intracellular free Ca2+ was identified by the calcium imaging technique as previously described [21] (link). Briefly, the culture medium was replaced with normal bath solution [140 mM NaCl, 5.0 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 5.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4)] containing Fluo-3 acetoxymethyl ester (2 μM, Invitrogen) and 0.1% pluronic F-127 (Invitrogen). After incubation for 40 min, the solution containing Fluo-3 acetoxymethyl ester was washed out with normal bath solution, and 1 mM CQ was applied to the cells to elicit calcium influx. For cases with KRGE and Rg3, these compounds were preincubated for 5 min prior to CQ application. The fluorescent intensities were measured at 488 nm with interval of 1.5 s under an inverted microscope (ECLIPSE Ti-U, Nikon, Tokyo, Japan). Intracellular Ca2+ changes were expressed as F/F0 ratios, where F0 was the initial fluorescence intensity. Image analysis was performed using ImageJ (NIH, Bethesda, MD, USA) with custom-made scripts for automatic cell counting, florescence intensity calculation and ratiometric image production.
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