MitoTracker Red CMXRos is a passively diffusing permeable probe containing mild thiol-reactive chloromethyl that accumulates in active mitochondria. The determined intensity of red fluorescence produced by O2•− by cells incubated with the MitoTracker Red CMXRos biosensor (200 nM MitoTracker Red CMXRos) in the dark for 10 min before the actual cell measurement. We detected the changes in ROS using confocal spinning disk microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) equipped with 100 × objective lens (Plan-Fluor × 100/1.45 Oil, Zeiss), a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, AOTF-based laser merge module for laser line 405, 445, 473, 488, 515, and 561 nm (Visitron Systems), and a Nipkow-based confocal scanning unit (CSUX1, Yokogawa Electric Corporation). Cells A375 with MitoTracker Red CMXRos were alternately excited with 579 nm laser lines, and emissions were acquired at 599 nm using a charged CCD camera (Cool SNAP-HQ, Photometrics, Tucson, AZ, USA). Z-stacks of channel in 0.2 µm increments were recorded. The VisiView acquisition software (Universal Imaging, Visitron Systems) was used to acquire the imaging data. Modified protocol according to Madreiter-Sokolowski et al.102 (link).
Visiview acquisition software
Manufactured by Visitron
VisiView acquisition software is a tool used to capture and manage data from various types of microscopes and imaging devices. It provides a platform for controlling instrument parameters, acquiring images, and organizing experimental data.
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5 protocols using visiview acquisition software
1
Quantifying Mitochondrial ROS Using MitoTracker Red
2
Imaging ER and Mitochondria Colocalization
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ER was labeled with D1ER and mitochondria stained with TMRM (50 nM). Cells were imaged on a confocal spinning disk microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) equipped with 100× objective lens (Plan-Fluor x100/1.45 Oil, Zeiss), a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, AOTF-based laser merge module for laser line 405, 445, 473, 488, 561, and 561 nm (Visitron Systems) and a Nipkow-based confocal scanning unit (CSU-X1, Yokogawa Electric corporation). The D1ER and TMRM were alternately excited with 488 and 561 nm laser lines, respectively, and emissions were acquired at 530 and 600 nm using a charged CCD camera (CoolSNAP-HQ, Photometrics, Tucson, AZ, USA). Z-stacks of both channels in 0.2 µm increments were recorded. VisiView acquisition software (Universal Imaging, Visitron Systems) was used to acquire the imaging data. Images were blindly deconvoluted with NIS-elements v5.1 (Nikon, Vienna, Austria). The colocalization was determined on a single-cell level using ImageJ and the plugin coloc2. The Pearson coefficient was calculated.
3
Visualizing MICU1 Localization in HeLa Cells
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Hela cells were transfected with tagged MICU1 constructs (MICU1-WT/MICU1-K/MICU1-F). After 2 days, cells were stained for 10 min with 200 nM MitoTracker Red CMXRos and imaged directly. High-resolution images of cells were recorded by using a confocal spinning disk microscope (Axio Observer.Z1 from Zeiss) equipped with × 100 objective lens (Plan-Fluor × 100/1.45 Oil, Zeiss), a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, AOTF-based laser merge module for laser line 405, 445, 473, 488, 561, and 561 nm (Visitron Systems), and a Nipkow-based confocal scanning unit (CSU-X1, Yokogawa Electric Corporation). The MICU1-YFP constructs and Mitotracker Red CMXRos were excited with 488 and 561 laser lines, respectively, and emission was acquired with a charged CCD camera (CoolSNAP-HQ, Photometrics, Tucson, AZ, USA). The software VisiView acquisition software (Universal Imaging, Visitron Systems) was used to acquire the imaging data. Images were background corrected with an ImageJ-Plugin (Mosaic Suite, background substractor).
4
Imaging of ER-Mitochondria Colocalization
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D1ER-overexpressing HeLa cells were stained for 10 min with 200 nM MitoTracker® Red CMXRos and imaged directly. High-resolution images of cells were recorded by using a confocal spinning disk microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) equipped with 100x objective lens (Plan-Fluor x100/1.45 Oil, Zeiss), a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, AOTF-based laser merge module for laser line 405, 445, 473, 488, 561, and 561 nm (Visitron Systems) and a Nipkow-based confocal scanning unit (CSU-X1, Yokogawa Electric corporation). The D1ER and Mitotracker® Red CMXRos were alternately excited with 488 and 561 nm laser lines, respectively, and emissions were acquired at 353 and 600 nm using a charged CCD camera (CoolSNAP-HQ, Photometrics, Tucson, AZ, USA). Z-stacks of both channels in 0.2 μm increments were recorded. The software VisiView acquisition software (Universal Imaging, Visitron Systems) was used to acquire the imaging data. Images were blind deconvoluted with NIS-elements (Nikon, Austria). The colocalization was determined on a single cell level using ImageJ and the plugin coloc2. The Pearson coefficient and the Costes thresholded Manders coefficient were calculated.
5
Visualizing Mitochondrial-ER Interactions in PAEC Cells
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D1ER-containing adenovirus infected PAEC cells were stained for 10 min with 200 nM MitoTracker® Red CMXRos and imaged directly. High resolution images of cells were recorded by using a confocal spinning disk microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) equipped with 100x objective lens (Plan-Fluor x100/1.45 Oil, Zeiss), a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, AOTF-based laser merge module for laser line 405, 445, 473, 488, 561, and 561 nm (Visitron Systems) and a Nipkow-based confocal scanning unit (CSU-X1, Yokogawa Electric corporation). The D1ER and Mitotracker ® Red CMXRos were alternately excited with 488 and 561 nm laser lines, respectively, and emissions were acquired at 353 and 600 nm using a charged CCD camera (CoolSNAP-HQ, Photometrics, Tucson, AZ, USA). Z-stacks of both channels in 0.2 µm increments were recorded. The software VisiView acquisition software (Universal Imaging, Visitron Systems) was used to acquire the imaging data. Images were blind deconvoluted with NIS-elements (Nikon, Austria). The colocalization was determined on a single cell level using ImageJ and the plugin coloc2. The Pearson coefficient and the Costes tresholded Manders 1 or 2 coefficients were calculated. The MitoTracker® Red CMXRos channel was assigned to channel 1 and D1ER to channel 2.
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