MitoSOX™ Red (Molecular Probes), a fluorogenic dye for selective detection of ROS levels in the mitochondria of live cells was used. Briefly, cells were washed with fresh media, and then incubated in media containing MitoSOX Red (2 μM), for 30 min at 37 °C in dark conditions then subjected to fluorescence microscopy at an excitation of 510 nm and an emission at 580 nm. An Olympus IX51 microscope equipped with a CCD camera (Hamamatsu Photonics) was used for acquisition of fluorescent images. The average fluorescent intensities (to correct for differences in cell number) were quantified using ImagePro Plus version 5.0 imaging software (Media Cybernetics, Rockville, MD).
Ix51 microscope
Manufactured by Hamamatsu Photonics
The IX51 microscope is a high-performance optical microscope designed for a variety of scientific and research applications. It features a stable, vibration-resistant frame and offers various illumination methods, including bright-field, phase contrast, and fluorescence imaging. The IX51 is capable of providing high-quality, detailed images of samples at different magnification levels.
Automatically generated - may contain errors
Lab products found in correlation
Ccd camera, by Hamamatsu Photonics (7 mentions)
Imagepro plus version 5.0 imaging software, by Media Cybernetics (5 mentions)
Mitosox red mitochondrial ros indicator, by Thermo Fisher Scientific (2 mentions)
C4742 95, by Hamamatsu Photonics (2 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Image pro plus version 5, by Media Cybernetics (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
C11440, by Olympus (1 mentions)
Eclipse te2000 u microscope, by Hamamatsu Photonics (1 mentions)
Prolong gold antifade reagent, by Cell Signaling Technology (1 mentions)
Prism 5, by GraphPad (1 mentions)
Daf fm diacetate, by Thermo Fisher Scientific (1 mentions)
9 protocols using ix51 microscope
1
Mitochondrial ROS Detection with MitoSOX
2
Mitochondrial ROS and Membrane Potential Imaging
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MitoSOX™ Red mitochondrial ROS indicator (Molecular Probes, Grand Island, NY) a fluorogenic dye for detection of ROS in the mitochondria of live cells was used. Mitochondrial membrane potential was determined using TMRM (tetramethylrhodamine methyl ester perchlorate, Molecular Probes, Eugene, OR). Briefly, cells were washed with fresh media, incubated in media containing MitoSOX Red (5 μM) or TMRM (50 nM), for 30 min at 37 °C in dark conditions, then subjected to fluorescence microscopy using an excitation of 510 nm and an emission at 580 nm (for MitoSOX) or an excitation of 548 nm and an emission at 575 nm (for TMRM). An Olympus IX51 microscope equipped with a CCD camera (Hamamatsu Photonics) was used for acquisition of fluorescent images. The average fluorescent intensities (to correct for differences in cell number) were quantified using ImagePro Plus version 5.0 imaging software (Media Cybernetics).
3
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential was determined using tetramethylrhodamine methyl ester perchlorate, TMRM (Molecular Probes, Eugene, OR). Briefly, cells were washed with fresh media, and incubated in media containing TMRM (50 nM), for 30 min at 37 °C in dark conditions then subjected to fluorescence microscopy at an excitation of 510 nm and an emission at 580 nm. An Olympus IX51 microscope equipped with a CCD camera (Hamamatsu Photonics) was used for acquisition of fluorescent images. The average fluorescent intensities (to correct for differences in cell number) were quantified using ImagePro Plus version 5.0 imaging software (Media Cybernetics).
4
Quantifying Mitochondrial Superoxide Levels
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MitoSOX™ Red mitochondrial superoxide indicator (Molecular Probes, Grand Island, NY) a fluorogenic dye for selective detection of superoxide in the mitochondria of live cells was used. Briefly, cells were washed with fresh media, and then incubated in media containing MitoSOX Red (2 µM), for 30 min at 37 °C in dark conditions then subjected to fluorescence microscopy at an excitation of 510 nm and an emission at 580 nm. An Olympus IX51 microscope equipped with a CCD camera (Hamamatsu Photonics) was used for acquisition of fluorescent images. The average fluorescent intensities (to correct for differences in cell number) were quantified using ImagePro Plus version 5.0 imaging software (Media Cybernetics).
5
Immunofluorescence Microscopy of HLMVEC
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HLMVEC were grown on cover glass till 100% confluent then exposed to the appropriate intervention. Cells were then fixed with 4% paraformaldehyde (Thermo Fisher Scientific) for 30 min, permeabilized with 100% cold methanol at -20 °C for 5 min. Cells then blocked with 1% BSA for 1 h, and incubated with first antibody overnight at 4 °C overnight, then the secondary antibody at room temperature for 1 h. Finally, cells were mounted on microscope slides using Prolong Gold Anti Fade Reagent (Cell Signaling Technologies®). Immunofluorescent images were observed with a Nikon Eclipse TE2000-U microscope, with Hamamatsu digital camera C11440, and Olympus IX51 microscope with Hamamatsu digital camera C4742-95. The images were analyzed with ImagePro Plus 7.0 to evaluate the colocalization of fluorescent.
6
Metaphase Chromosome Spread Analysis
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Cells were seeded in 6‐well plates at 1 × 105 cells/well, treated as required and then washed three times with PBS, followed by addition of fresh media containing 200 ng/ml nocodazole for 16 h. Cells were harvested and swollen with 75 mM KCl for 20 min at 37 °C before being fixed with methanol:acetic acid (3:1). The fixing step was repeated two times. Cells were then dropped onto pre‐hydrated glass slides and air‐dried overnight before coverslips were mounted onto the slides with Vectashield containing DAPI. Images of metaphase spreads were acquired with a 60× objective using an Olympus IX51 microscope equipped with a Hamamatsu camera (C4742‐95). Images were processed using ImageJ/Fiji software 73 , and visible chromatid breaks were scored. Statistical analysis was performed using Prism 5 (GraphPad Software).
7
Mitochondrial ROS Detection in Live Cells
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MitoSOX™ Red mitochondrial ROS indicator (Molecular Probes, Grand Island, NY) a fluorogenic dye for detection of ROS in the mitochondria of live cells was used. Briefly, cells after treatment were washed with fresh serum free medium, incubated with MitoSOX Red (5 μM), for 30 min at 37 °C in dark conditions, then subjected to fluorescence microscopy using an excitation of 510 nm and an emission at 580 nm (for MitoSOX). An Olympus IX51 microscope equipped with a CCD camera (Hamamatsu Photonics) was used for acquisition of fluorescent images. The average fluorescent intensities (to correct for differences in cell number) were quantified using ImagePro Plus version 5.0 imaging software (Media Cybernetics).
8
Quantifying NO levels in Cultured PAECS
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NO levels in cultured PAECS were determined by using DAF-FM diacetate (ThermoFisher, Waltham, MA), a cell-permeable fluorescent dye, according to the product’s instruction. Briefly, cells were aspirated, rinsed in PBS and cultured in media with DAF-FM diacetate (5 μM) for 30 min in the dark at 37 °C, then subjected to fluorescence microscopy. Fluorescent images were taken by using an Olympus IX51 microscope equipped with a CCD camera (Hamamatsu Photonics). We quantified the average fluorescent intensities by using ImagePro Plus version 5.0 imaging software (Media Cybernetics).
9
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential was determined using TMRM (tetramethylrhodamine methyl ester perchlorate, Molecular Probes, Eugene, OR). Briefly, after each experiment, cells were washed with fresh media, incubated in media containing TMRM (50 nM), for 30 min at 37 °C in dark conditions, then subjected to fluorescence microscopy using an excitation of 548 nm and an emission at 575 nm. An Olympus IX51 microscope equipped with a CCD camera (Hamamatsu Photonics) was used for acquisition of fluorescent images and the average fluorescent intensities were quantified using ImagePro Plus version 5.0 imaging software (Media Cybernetics).
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