Mitored dye

Manufactured by Merck Group

Sourced in Poland

MitoRed is a fluorescent dye that selectively stains mitochondria in live cells. It is a cationic dye that accumulates in the mitochondrial matrix due to the negative membrane potential. MitoRed exhibits increased fluorescence upon binding to mitochondria, allowing for visualization and analysis of mitochondrial structure and function.

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Lab products found in correlation

Er tracker green, by Thermo Fisher Scientific (2 mentions) Faramount aq mounting medium, by Agilent Technologies (2 mentions) Observer z1 confocal spinning disc v 2, by Zeiss (2 mentions) Phalloidin atto 488, by Merck Group (2 mentions) Leica tcs spe, by Leica Microsystems (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Atto 488 labeled phalloidin, by Merck Group (1 mentions) Triton x 100, by Merck Group (1 mentions) Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)

4 protocols using mitored dye

Multimodal Cellular Imaging Protocol

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A confocal microscope (Observer Z1 Confocal Spinning Disc V.2 Zeiss) was used to visualize cell organelles.
The endoplasmic reticulum was stained with 1 µM ER-Tracker Green (E34251, Invitrogen, Thermo Fisher Scientific, Warsaw, Poland). The cell medium was changed to Hankʼs Balanced Salt Solution with calcium and magnesium containing 1 µM ER-Tracker Green. Cells were incubated for 30 min at 37 °C. Then, cells were fixed with 4% PFA (Sigma-Aldrich/Merck, Poznan, Poland) as described above. Cell nuclei were stained with DAPI (Faramount Aq Mounting Medium, Dako, Santa Clara, CA, USA).
Mitochondria were stained with MitoRed dye (53271, Sigma-Aldrich/Merck, Poznań, Polska) (1:1000 in cell medium) on viable cells for 30 min. The cells were then fixed with PFA (Sigma-Aldrich/Merck, Poznan, Poland) as described above. Cells were permeabilized with 0.1% Triton X-100 (93443, Sigma-Aldrich/Merck, Poznan, Poland) solution for 15 min. The cytoskeleton was stained using atto-488-labeled phalloidin (49409, Sigma-Aldrich/Merck, Poznan, Poland) (1:800 in PBS) for 40 min in the dark at RT. The cells were washed three times in PBS. Cell nuclei were stained with DAPI (Faramount Aq Mounting Medium, Dako, Santa Clara, CA, USA).

Kowalczuk A., Marycz K., Kornicka-Garbowska K., Kornicka J., Bujalska-Zadrożny M, & Groborz S. (2022). Cannabidiol (CBD) Protects Adipose-Derived Mesenchymal Stem Cells (ASCs) against Endoplasmic Reticulum Stress Development and Its Complications. International Journal of Environmental Research and Public Health, 19(17), 10864.

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Multicolor Cellular Imaging of Subcellular Structures

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A confocal microscope (Observer Z1 Confocal Spinning Disc V.2 Zeiss) was used for the visualization of the cell nucleus, mitochondria, cytoskeleton and the endoplasmic reticulum. The endoplasmic reticulum was stained with 1 µM ER-Tracker™ Green (Invitrogen™, E34251, Thermo Fisher Scientific, Warsaw, Poland). For this, cell medium was changed to Hank’s Balanced Salt Solution with calcium and magnesium containing 1 µM ER-Tracker™ Green. After 30 min (min) of incubation at 37 °C, cells were fixed with cold 4% paraformaldehyde for 30 min. Mitochondria were stained with 100 nM MitoRed dye (Sigma-Aldrich, 53271, Poznan, Poland) on viable cells for 30 min in the dark at 37 °C. Medium containing the MitoRed dye was removed, and cells were washed three times with PBS. Cells were fixed with 4% PFA as described above and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, 93443, Poznan, Poland) solution for 20 min. The cytoskeleton was stained using atto-488-labeled PI (Sigma-Aldrich, 49409, Poznan, Poland) (1:800 in PBS) for 45 min in the dark at RT. Cell nuclei were stained with DAPI (Invitrogen™, Warsaw, Poland), following the instructions of the manufacturer (Faramount Aq Mounting Medium, Dako).

Kowalczuk A., Marycz K., Kornicka J., Groborz S., Meissner J, & Mularczyk M. (2023). Tetrahydrocannabivarin (THCV) Protects Adipose-Derived Mesenchymal Stem Cells (ASC) against Endoplasmic Reticulum Stress Development and Reduces Inflammation during Adipogenesis. International Journal of Molecular Sciences, 24(8), 7120.

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Nanoparticle-mediated Cytotoxicity Assessment

Cited 2 times

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The potential cytotoxic effect of nHAP/IO and nHAP/IO@miR-21/124 was tested based on ultrastructural features of cells, and mitochondrial metabolism determined using Alamar Blue assay. The metabolic activity of cells measured with Alamar Blue assay was evaluated after 4 days of the experiment, using the protocol established previously and according to the manufacturer's instructions.28 (link) For confocal imaging, cultures were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. The mitochondria network structure was determined after its staining with MitoRed dye (Sigma-Aldrich, Munich, Germany). Actin cytoskeleton was visualised using a solution of phalloidin-Atto 488 (Sigma-Aldrich, Munich, Germany) prepared at a concentration of 1:800 in phosphate-buffered saline pH 7.4. Mitochondria and cytoskeleton staining were described in detail elsewhere.29 (link) The specimens were preserved using Mounting Medium with DAPI ([4ʹ,6-diamidino-2-phenylindole], Thermo Fisher Scientific, Waltham, MA, USA) for nuclei counterstain. The specimens imaging was done with confocal fluorescence microscopy (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany) under 630× magnification. The analysis was also supported by measuring mRNA-coding anti- and pro-apoptotic molecules, Bcl-2 and Bax, respectively. The description of transcripts determination is described below.

Marycz K., Śmieszek A., Kornicka-Garbowska K., Pielok A., Janeczek M., Lipińska A., Nikodem A., Filipiak J., Sobierajska P., Nedelec J.M, & Wiglusz R.J. (2021). Novel Nanohydroxyapatite (nHAp)-Based Scaffold Doped with Iron Oxide Nanoparticles (IO), Functionalized with Small Non-Coding RNA (miR-21/124) Modulates Expression of Runt-Related Transcriptional Factor 2 and Osteopontin, Promoting Regeneration of Osteoporotic Bone in Bilateral Cranial Defects in a Senescence-Accelerated Mouse Model (SAM/P6). PART 2. International Journal of Nanomedicine, 16, 6049-6065.

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Visualization of ASC and HPC Morphology

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The morphology of native ASCs and HPCs was assessed using fluorescent staining. For the mitochondrial network visualization, the mitochondria were stained with MitoRed dye (Sigma-Aldrich/Merck, Poznan, Poland) prepared in a culture medium (1:1000). Phalloidin-Atto 488 (Sigma-Aldrich/Merck, Poznan, Poland) was used to visualize the actin cytoskeleton. The nuclei were stained with 4′,6-Diamidine-2′-phenylindole dihydrochloride, and the coverslips were mounted onto glass slides with ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific, Warsaw, Poland). The cells were observed and photographed using a confocal microscope under 630× magnification (Leica TCSSPE, Leica Microsystems, KAWA.SKA Sp. z o. o., Zalesie Górne, Poland). The obtained images were processed using Fiji software (ImageJ 1.52n, Wayne Rasband, National Institute of Health, Bethesda, MD, USA).

Pielok A., Kępska M., Steczkiewicz Z., Grobosz S., Bourebaba L, & Marycz K. (2023). Equine Hoof Progenitor Cells Display Increased Mitochondrial Metabolism and Adaptive Potential to a Highly Pro-Inflammatory Microenvironment. International Journal of Molecular Sciences, 24(14), 11446.

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