Mitotrackertm red cmxros

Manufactured by Thermo Fisher Scientific

Sourced in United States

MitoTrackerTM Red CMXRos is a fluorescent dye that can be used to stain and visualize mitochondria in live cells. It is a cell-permeant dye that accumulates in active mitochondria, providing a way to monitor mitochondrial membrane potential and morphology.

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29 protocols using mitotrackertm red cmxros

Mitochondrial Imaging of FRDA BMSCs

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10,000 Au8-pXs labeled and unlabeled BMSCs from FRDA patients were stained for mitochondria into 4-well chamber slides with MitoTracker TM Red CMXRos (Thermo Fisher Scientific).
Cells were treated with 50nM MitoTracker TM Red CMXRos in cell culture medium without serum for 30 minutes at 37C, and then fixed with 4% paraformaldehyde (PFA) (Thermo Fisher Scientific) for 10 minutes. Leica SP8 confocal microscope was used to acquire images. Au8-pXs fluorescence was detected setting the emission range between 500 and 540 nm under 405 nm excitation. Each cell group was evaluated in five independent experiments. Colocalization of mitochondria and gold clusters superstructures was evaluated by EZColocalization plugin of Image J, identifying single cells through hand drawn ROIs. The result was represented as heatmap image between 0 and 250.

Villa C., Legato M., Umbach A., Riganti C., Jones R., Martini B., Boido M., Medana C., Facchinetti I., Barni D., Pinto M., Arguello T., Belicchi M., Fagiolari G., Liaci C., Moggio M., Ruffo R., Moraes C.T., Monguzzi A., Merlo G.R, & Torrente Y. (2021). Treatment with ROS detoxifying gold quantum clusters alleviates the functional decline in a mouse model of Friedreich ataxia. Science translational medicine, 13(607).

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Immunofluorescence Staining of Aneuploid Cells

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HCT116 and RPE1 cells and their aneuploid derivatives were seeded to a 96-well plate (HCT116: 10⁴ cells per well, RPE1: 10³ cells per well) and incubated at 37 °C and 5% CO₂ on the day before experiments. For cell fixation, a 3% formaldehyde solution was used for 15 min at room temperature. Next, cells were permeabilized with 0.1% Triton for 20 min at room temperature. Then, cells were preblocked with 3% bovine serum for 30 min at room temperature. Primary antibodies were diluted in blocking solution (Supplementary table 2) and incubated overnight at 4 °C. The next day, cells were washed and secondary antibodies were added in a concentration of 1 mg per ml followed by incubation for 1 h in the dark at room temperature. SYTOX® green (Invitrogen) or DAPI (Invitrogen) were used to localize the nucleus. For cytoplasmic staining, the HCS Cell Mask (H327 12 Component, 701618, Invitrogen) was applied. The cells were covered with SlowFade Gold Antifade (Invitrogen). To localize mitochondria, MitoTracker^TM Red CMXRos (M7512, Invitrogen) was used (100 nM for 45-h incubation at 37 °C). For lysosome localization, we also used LysoTracker^TM Red DND-99 (L7528, Thermofisher Scientific) according to the manufacturer’s protocol. Information regarding the used antibodies can be found in Supplementary table 2.

Krivega M., Stiefel C.M., Karbassi S., Andersen L.L., Chunduri N.K., Donnelly N., Pichlmair A, & Storchová Z. (2021). Genotoxic stress in constitutive trisomies induces autophagy and the innate immune response via the cGAS-STING pathway. Communications Biology, 4, 831.

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Immunofluorescence Analysis of DNA Damage and Mitochondrial Localization

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For γH2AX foci analysis, cells were first seeded on sterile glass coverslips in 12-well plates. Cells were fixed and permeabilized as previously described [11 (link)], incubated with 1:400 diluted polyclonal anti-γH2AX antibody (Abcam) and detected with secondary Alex Fluor^® 488-conjugated goat anti-mouse IgG (Life Technologies). For EdU staining, cells were pulse labeled with 10 μM EdU (Invitrogen) prior to cell fixation. EdU incorporation was detected with the Click-iT^® EdU AlexaFluor^® 594 imaging kit (Invitrogen). Digital images were captured using a LEICA TCS SP5 confocal microscopy system and analyzed using Fiji software.
To visualize DCTPP1, HAP1 WT and DCTPP1-KO, cells were grown on sterile glass coverslips. For mitochondrial labeling, cells were incubated with 250 nM of MitoTrackerTM Red CMX Ros (Invitrogen) for 30 min at 37 °C. Fixation, permeabilization and blocking were performed as previously described. Cell were incubated with anti-DCTPP1 at a 1:1000 dilution for 1 h, washed and incubated with Alexa Fluor^® 488-conjugated anti-rabbit secondary antibody. Digital images were captured using a LEICA TCS SP5 confocal microscopy system.

Martínez-Arribas B., Requena C.E., Pérez-Moreno G., Ruíz-Pérez L.M., Vidal A.E, & González-Pacanowska D. (2019). DCTPP1 prevents a mutator phenotype through the modulation of dCTP, dTTP and dUTP pools. Cellular and Molecular Life Sciences, 77(8), 1645-1660.

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Visualizing Mitochondrial Morphology in Cells

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Cells were grown at low density on coverslips. For visualization of mitochondrial morphology, cells were incubated with 200 nM MitoTracker ^TM Red CMXRos (Invitrogen, Eugene, OR) for 20 min. Cells were fixed in cold 4% paraformaldehyde and permeabilized and blocked with 0.3% Triton X-100 and 5% goat serum in PBS for 1 h at room temperature. Cells were incubated with the following primary antibodies diluted in 5% goat serum in PBS at 4 °C overnight: rabbit anti-TOM20 (1:300, Cell Signaling Technology, Danvers, MA, USA), chicken anti-MAP2 (1:1000, Invitrogen, Eugene, OR, USA), rabbit anti-Willin/FRMD6 (1:100, Cell Signaling Technology, Danvers, MA, USA). Corresponding fluorescent secondary antibodies (Alexa Fluor^TM 488, 568, 594, 647, 1:1000, Invitrogen, Eugene, OR, USA), were diluted in blocking buffer and incubated for 1 h at room temperature. Coverslips were mounted in ProLong^TM Diamond Antifade Mountant (Invitrogen, Eugene, OR, USA) and imaged with a Leica TCS SP8 confocal microscope using a 63× oil immersion objective or a Leica DM5500B epifluorescence microscope (Mannheim, Germany) using a 40× oil immersion objective.

Chen D., Yu W., Aitken L, & Gunn-Moore F. (2022). Willin/FRMD6 Mediates Mitochondrial Dysfunction Relevant to Neuronal Aβ Toxicity. Cells, 11(19), 3140.

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Visualizing Mitochondrial Dynamics in Oocytes

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To observe the mitochondrial distribution in oocytes before and after vitrification, live oocytes were washed two times with M2 media and stained with MitoTracker^TM Red CMXRos (200 nmol/L, M7512, Invitrogen) at 37 °C in 5% CO₂ for 15 min. They were then treated with NucBlue^TM Live Cell Stain ReadyProbes^TM reagent (R37605, Invitrogen) for 15 min in an incubator. Oocytes were washed two times with M2 media and transferred to a glass-bottom confocal dish (P35G-1.0-14-C, MatTek, Ashland, MA, USA). Live images of mitochondria in oocytes (red color) were obtained directly using a confocal microscope (Zeiss LSM880), and analyzed using ZEN software (ZEN 2012, Blue edition, Carl Zeiss) after red was converted to pseudo-color (green) for better visualization. For quantification of the mitochondrial distribution, fluorescence intensity profiles were measured using the ImageJ software (v1.48, NIH, Bethesda, Maryland, USA).

Lee J.H., Park J.K., Yoon S.Y., Park E.A., Jun J.H., Lim H.J., Kim J, & Song H. (2021). Advanced Maternal Age Deteriorates the Developmental Competence of Vitrified Oocytes in Mice. Cells, 10(6), 1563.

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Mitochondrial Staining and Analysis

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Briefly, fixed cells were incubated with 200 nM of MitoTracker^TM Green FM (Invitrogen) or 500 nM of MitoTracker^TM Red CMXRos (Invitrogen, Waltham, MA, USA) in DPBS for 15 min at 37 °C. After washing with PBS, nuclei were stained with DAPI for 5 min, and fluorescence images were captured using a fluorescence microscope. The fluorescence intensity for mitochondrial mass and membrane potential were calculated using ImageJ (version 1.54d).

Hong S., Park S.K., Lee J., Park S.H., Kim Y.S., Park J.H., Yu S, & Lee Y.G. (2023). Patulin Ameliorates Hypertrophied Lipid Accumulation and Lipopolysaccharide-Induced Inflammatory Response by Modulating Mitochondrial Respiration. Antioxidants, 12(9), 1750.

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Visualizing Lipid and Mitochondrial Changes in Palmitic Acid-Treated HepG2 Cells

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HepG2 cells were seeded and pre-treated with either 20 or 40 μM of TM5441 for 4 hours prior to 400 μM palmitic acid (PA) stimulation for 24 hours. Neutral lipid was detected with BODIPY^TM 493/503 (Invitrogen, Carlsbad, CA, USA) and nucleus was subsequently stained with DAPI (Invitrogen, Carlsbad, CA, USA). With the same experimental condition, mitochondria was stained using Mitotracker^TM Red CMXRos (Invitrogen, Carlsbad, CA, USA). Following staining, the cells was fixed using 4% paraformaldehyde. The immunofluorescence stained cells were visualized using confocal microscopy (Carl Zeiss, Gottingen, Germany).

Lee S.M., Dorotea D., Jung I., Nakabayashi T., Miyata T, & Ha H. (2017). TM5441, a plasminogen activator inhibitor-1 inhibitor, protects against high fat diet-induced non-alcoholic fatty liver disease. Oncotarget, 8(52), 89746-89760.

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Mitochondrial Staining and Quantification

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The cells were fixed with a 3.7% formaldehyde solution. After washing with PBS, fixed cells were incubated with 500 nM of MitoTracker^TM Red CMXRos (Invitrogen, Waltham, MA, USA) in PBS for 30 min at 37 °C. After rinsing with PBS thrice, cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min, and fluorescence images of the cells were captured using an Olympus DP71 fluorescence microscope (Olympus, Tokyo, Japan). The fluorescence intensity was measured in randomly selected regions and quantitatively analyzed using ImageJ software (version 1.54d).

Yu S., Lee H.M., Lee J., Hwang J.T., Choi H.K, & Lee Y.G. (2024). Pennogenin 3-O-β-Chacotrioside Attenuates Hypertrophied Lipid Accumulation by Enhancing Mitochondrial Oxidative Capacity. International Journal of Molecular Sciences, 25(5), 2970.

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Mitochondrial Morphology Visualization

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Mitochondrial morphology in MEF cells and patient fibroblasts was visualized using MitoTracker^TM Red CMXRos (Invitrogen, United States). The cells were seeded onto sterile cover slips the day prior to the experiment. Cells were incubated in 200 nM MitoTracker^TM Red CMXRos diluted in fresh DMEM for 30 min at 37°C. For SO₃^2– treatments, the respective SO₃^2– concentration was added to the medium 30 min prior to the MitoTracker and incubated at 37°C. Afterward, the cells were washed carefully with PBS before fixation in 4% PFA for 15 min at 4°C. Remaining PFA was removed by three washing steps with PBS (3 × 5 min). Finally, cover slips were mounted on slides with Mowiol/DABCO (Carl Roth). Images were acquired using a Nikon A1 confocal laser scanning microscope. Images were processed using ImageJ software.

Mellis A.T., Roeper J., Misko A.L., Kohl J, & Schwarz G. (2021). Sulfite Alters the Mitochondrial Network in Molybdenum Cofactor Deficiency. Frontiers in Genetics, 11, 594828.

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Mitochondrial Dysfunction and Oxidative Stress Protocol

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MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), MPP⁺ (1-methyl-4-phenylpyridium), MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide), rotenone, antimycin A, CCCP (carbonyl cyanide 3-chlorophenylhydrazone), oligomycin, and corn oil were obtained from Sigma-Aldrich (St. Louis, MO, USA). Evernic acid (EA) was purchased from Santa Cruz Technology (Santa Cruz, CA, USA). DCF-DA (2′-7′-di-chlorofluorescein diacetate), MitoTracker^TM Red CMXRos, Alexa Flour 488, Alexa Flour 568, and DAPI (4’,6-diamidino-2-phenylindole) were from Invitrogen (Carlsbad, CA, USA), and Western blot detection reagent (ECL solution) was purchased from Advansta (San Jose, CA, USA).

Lee S., Suh Y.J., Yang S., Hong D.G., Ishigami A., Kim H., Hur J.S., Chang S.C, & Lee J. (2021). Neuroprotective and Anti-Inflammatory Effects of Evernic Acid in an MPTP-Induced Parkinson’s Disease Model. International Journal of Molecular Sciences, 22(4), 2098.

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