Mitochondrial dysfunction was measured by MitoTracker Red CMXRos (Life Technologies). At 2, 4 or 8 weeks of differentiation, BrainSpheres were plated in 24-well-plates (500 μl), and exposed to different concentrations of rotenone (0, 0.1, 1, 10, 25, 50, 100 μM) for 12, 24 and 48 h. Thirty minutes before the end of exposure, 1 μl of MitoTracker Red CMXRos was added to the medium. Cells were incubated for 30 min at 37 °C, 5% CO2. After the incubation, 400 μl of medium was removed and 500 μl of 4% paraformaldehyde solution was added and incubated for 20 min at room temperature. After fixation, paraformaldehyde was removed and spheroids were washed twice with PBS. The Shandon Immuno-Mount (ThermoFisher Scientific) was used to mount the spheroids onto microscope cover slides (Fisher Scientific). An Olympus BX60 fluorescence microscope was used to image the MitoTracker fluorescence and processed with Image-pro Plus 7.0 (Media Cybernetics). The fluorescence was quantified using ImageJ software from NIH (https://imagej.nih.gov/ij/ ) and normalized to the size of the aggregates. To determine statistical significance, one-way ANOVA was performed with post-hoc Bonferroni test. All data given are the means ± SD of three to seven independent experiments performed with 5 technical replicates.
Microscope cover slides
Manufactured by Thermo Fisher Scientific
Sourced in United States
Microscope cover slides are thin, transparent plates made of glass or plastic that are used to cover and protect specimens during microscopic examination. They provide a flat, uniform surface that helps to keep the specimen in place and prevent it from drying out. Cover slides are an essential component of the microscopy process, facilitating the observation and analysis of small samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Shandon immuno mount, by Thermo Fisher Scientific (2 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (2 mentions)
Image pro plus 7, by Media Cybernetics (1 mentions)
Bx60 fluorescence microscope, by Media Cybernetics (1 mentions)
Sodium cacodylate buffer, by Merck Group (1 mentions)
Em 10 transmission electron microscope, by Zeiss (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
4 protocols using microscope cover slides
1
Mitochondrial Dysfunction in BrainSpheres
2
Immunofluorescence Microscopy of Cells
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Cells were seeded on microscope cover slides (Fisherbrand, Waltham, MA, USA) and incubated overnight. After the treatments, the cells were washed three times with PBS and fixed with 4% formaldehyde for 15 min. Then, the cells were permeabilized with 0.25% Triton X-100 in PBS for 10 min and blocked with goat serum for blocking (Boster, Pleasanton, CA, USA) for 1 h at room temperature. Cells were stained with primary antibodies for 10 h at 4 °C and then incubated with Alexa Fluor Plus 555 goat anti-rabbit IgG secondary antibodies (Invitrogen) at a dilution of 1:1000 for 2 h at RT. Images were taken on a Zeiss LSM 710 confocal microscope.
3
Mitochondrial Dysfunction in BrainSpheres
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Mitochondrial dysfunction was measured by MitoTracker Red CMXRos (Life Technologies, Carlsbad, CA, USA) following the protocol described in Pamies et al. (2018 (link)). Briefly, after 8 weeks of exposure to 0, 20 or 60 ng/ml of paroxetine, 10 BrainSpheres per condition were plated in 24-well-plates (500 μl). One microliter of MitoTracker Red CMXRos was added to the medium and incubated for 30 min at 37°C, 5% CO2. The BrainSpheres were then washed twice and fixed with 4% paraformaldehyde (PFA) for 1 h and washed again twice with PBS. The Shandon Immuno-Mount (Thermo Fisher Scientific, Waltham, MA, USA) was used to mount the spheroids onto microscope cover slides (Thermo Fisher Scientific, Waltham, MA, USA). Images were taken using a Olympus BX60. The fluorescence was quantified using ImageJ software1 and normalized to the size of the aggregates. To determine statistical significance, one-way ANOVA was performed with post hoc Bonferroni test. All data given are the means ± SD of three independent experiments performed with 10 technical replicates.
4
Ultrastructural Analysis of Dexamethasone-Treated Cells
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The cells were seeded in 24-well plates containing microscope cover slides (Thermo Fisher, Saarbrucken, Germany) at a density of 3 × 104/ml and in the presence of 1 µM DEX or ethanol solvent control for 24 h. Afterward, the cells were fixed with 2.5% glutaraldehyde (Sigma‒Aldrich, Taufkirchen, Germany) in 0.1 mM sodium-cacodylate buffer (Sigma‒Aldrich). The samples were stained with a negatively charged 1% aqueous phosphotungstic acid solution or a 2% acetate solution (both from Sigma‒Aldrich), which were dropped into a 200 μm mesh-size pioloform-coated copper grid or a microscope carbon-coated grid using a micropipette. The samples were analyzed under a Zeiss EM10 transmission electron microscope (Carl Zeiss, Oberkochen, Germany) at 80 kV and ×6000 magnification.
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