A second mitochondrial fusion assay was developed to take advantage of photoactivatable gfp pagfp. Request pdf labeling mitochondria with mitotracker dyes introduction membranepotentialdependent dyes such as rhodamine 123. Their gtpase domain uses energy for contraction and sequeezing the membranes next to each other so that fusing can occur. Mitochondrial biogenesis allows neurons to meet changing energy loads and to redistribute mitochondria throughout all compartments of the neuron. Mitochondrial imaging in live or fixed tissues using a luminescent. Once their mitochondria are labeled, the cells can be treated with aldehydebased fixatives to allow further processing of the sample. Mitochondria were stained with mitotracker deep red and 3dimensional reconstructions of the cells were performed from a series of zstacks. Mitochondrial structure, function and dynamics are temporally. Mitotracker is a commercially available fluorescent dye invitrogenmolecular probes that, like the aforementioned dyes, labels mitochondria within live cells utilizing the mitochondrial membrane.
Mitochondrial morphological features are associated with. This protocol presents methods for labeling mitochondria in both live and fixed cells using several different fluorescent molecules. Mitophagy is an important mechanism in mitochondrial quality control through autophagic clearance of damaged mitochondria and has been considered to assume protective roles against some diseases, especially neurodegeneration, including parkinsons disease haelterman et al. Labeling mitochondria with fluorescent dyes for imaging. Eukaryote cells, meaning animal cells more advanced organisms are the type of cells that have them, and they need mitochondria to function properly. Mitotracker red cmxros, mitotracker red 580 and mitotracker deep red 633 are all well suited for multicolor labeling experiments because their red fluorescence is well resolved from the green fluorescence of other probes. If mitochondrial labeling of fixed cells is desired. Full text pdf to view this item, select one of the options below. Mitotracker thermo fisher scientific is a similar label that has an additional chloromethyl moiety, which reacts with thiols in mitochondria keeping mitotracker associated with mitochondria even after fixation poot et al. When combined with labeling of the mitochondria by immunofluorescence or mitotracker loading, mtrip allows classification of the organelles. The products described in this manual are covered by one or more limited use label. Mitotracker dyes are fluorescent mitochondrial markers that. To improve photostability of mitochondrial staining, the mitotracker red dye cmxrosamine. The dye will stain live cells but is not wellretained after aldehyde fixation.
High quality imaging of mitochondria is of great interest for medical applications. Labelfree optical imaging of mitochondria in live cells. Methods to assess subcellular compartments of muscle in c. Colabeling with mitotracker orange or mitotracker green revealed almost complete overlap of the mitotracker dyes with the structures stained with r123 and tmre fig. Many of these dyes also allow crude assessment of mitochondrial functionality in vivo because they accumulate in mitochondria based upon the mitochondrial membrane potential. Mitotracker dyes are cell permeable probes that contain a mildly thiolreactive chloromethyl moiety for mitochondrial labeling. This protocol describes the labeling of mitochondria in live and fixed cells with mitotracker dyes.
A method for measuring mitochondrial mass and activity. The hr2 domains interacts and grasps the hr2 domain of another mitochondria. To label mitochondria, cells are simply pattern of the mitotracker dyes. To understand the role of mitophagy in these diseases, measures to. Mitotracker and mitofluor mitochondrionselective probes. The process involves physical merging of both the outer and inner membranes of mitochondria. Combining cellbased hts of large chemical libraries with microscopy is. Static and dynamic experiments demonstrated the dependence of dye accumulation in the mitochondria and cytoplasm on mitochondrial potential in the presence of. There is a printable worksheet available for download here so you can take the quiz with pen and paper. T1 analysis of individual mitochondria via fluorescent immunolabeling with antitom22 antibodies. Mtdr mitotracker deep red, previously used in this study to label all polarized mitochondria, is wellretained in subsequently depolarized mitochondria supplementary fig. Mitotracker is a commercially available fluorescent dye invitrogenmolecular probes that, like the aforementioned dyes, labels mitochondria within live cells. In addition, it can be used in experiments in which multiple labeling diminishes mitochondrial function.
Mitotracker dyes are fluorescent mitochondrial markers that covalently bind free sulfhydryls. I try to stain mitochondria on my primary cells with mitotracker red cmxros recently. Similar membranepermeable probes for labeling of endoplasmic reticulum have also been developed. Jun 24, 2008 mitochondria, responsible for the energygenerating process essential for the cell metabolism, differ for the number, localization and activity in animal cells and tissues in relation to the energetic needs. Mitotracker dyes are also cationic fluorophores that accumulate electrophoretically into mitochondria in response to the highly negative mitochondrial membrane potential. Quantitative analysis of mitochondrial morphology and. Shown are representative images with a spacefilling overlay to emphasize the mitochondria. To label mitochondria, cells are simply incubated with mitotracker probes, which passively diffuse across the plasma membrane and accumulate in active mitochondria. Analysis of individual mitochondria via fluorescent. While some mitochondrial labeling may be achieved in fixed cells using mitotracker dyes, the signaltonoise ratios are not generally favorable and these dyes are recommended for use in live cells only. The labelling of mitochondria by irazolvemito was compared to other available imaging technologies including live cell staining by mitotracker.
The mitochondria are bout the size of a bacterial cell and are often peanutshaped. Nanosecondelectricpulses causemitochondrialmembrane. Mitochondrial function provides instructive signals for. We report the discovery of a fluorogenic dye, n1,n3di2aminidonaphthalen6yl propane1,3diamine, mitoblue, which selectively stains functional mitochondria while displaying low toxicity, bright blue emission, and high resistance to photobleaching.
In this work, we report the farfield optical imaging of mitochondria of live cells without the use of any label. I haved tried the concentration with a range of 60200nm and incubate for 30min. Mitotracker is a commercially available fluorescent dye invitrogenmolecular probes that, like the aforementioned dyes, labels mitochondria within live cells utilizing the mitochondrial membrane potential. Rhodamine 123, tetramethylrhodamine ethyl ester tmre, and tetramethylrhodamine methyl ester tmrm are membranepotentialsensitive, cationic fluorophores. Rhodamine 123 is specifi c for mitochondria labeling, but tmre, tmrm, and jc1 dyes labels endoplasmic reticulum to some extent chazotte 2009. When this cellpermeant probe enters an actively respiring cell, it is oxidized to mitotracker orange cmtmros and sequestered in the mitochondria, where it reacts with thiols on proteins and. These dyes provide an alternative method of visualizing mitochondrial network structure in live c.
Potential experimental pitfalls mitochondria staining mitochondria are stained in live and fixed cells by adding to the cell medium respectively mitotracker green fm invitrogen at a final concentration of 20nm for 15min, or mitotracker deep red fm invitrogen at 100nm for 30min. Together, these 10channels were used to form our hyperlabeled image. Request pdf mitotracker labeling in primary neuronal and astrocytic cultures. Invitrogen mitotracker green fm special packaging 20 x 50ug. Introduction membranepotentialdependent dyes such as rhodamine 123,tetramethylrhodamine methyl ester tmrm, and tetramethylrhodamine ethyl estertmre are useful as long as the mitochondrion maintains its negative membranepotential. Fluorescent dyes used to label mitochondria include the mitotracker probes, which are available conjugated to a wide variety of fluorochromes. A guide to fluorescent probes and labeling technologies. Although several key regulators and physiological stimuli have been identified to control mitochondrial fission and fusion, the role of mitochondrial morphology in the two processes remains to be determined. The most widely utilized, acridine orange, consists of the basic acridine nucleus with dimethylamino substituents located at the 3 and 6 positions of the trinuclear ring system as illustrated in figure 4a. Comparison of mitochondrial fluorescent dyes in stromal cells. Can anybody give me protocol for mitochondria staining using.
Flow cytometrybased assessment of mitophagy using mitotracker. However, unlike tmrm and rhodamine 123, mitotracker dyes possess a reactive chloromethyl group that forms a covalent bond with thiols on proteins and peptides, which traps. Mitotracker is a commercially available fluorescent dye invitrogenmolecular probes that, like the aforementioned dyes, labelsmitochondria within live cells utilizing the mitochondrial membrane. The bright particles in calceinstained cells are not dense enough to merge over the entire. Mitochondrial health requires frequent fusion of mitochondria and subsequent division fission. However, mitotracker is chemically reactive, linking to thiol groups in the mitochondria. The impact of alterations in mitochondrial membrane potential m and oxidant stress on mitotracker staining in mitochondria in cultured neurons and astrocytes has been investigated. N2 mitochondria are responsible for maintaining a variety of cellular functions. Mitotracker labeling in primary neuronal and astrocytic cultures. The intracellular reactions of our fixable mitochondrionselective dye, mitotracker orange cmh2tmros. Mitochondrionspecific, smallmolecular fluorescent dyes may be categorized into. Mitotracker green fm is greenfluorescent mitochondrial stain which appears to localize to mitochondria regardless of mitochondrial membrane potential. Mitotracker green fm reacts with the free thiol groups of cystein residues.
Confocal microscopy fluorophores for confocal microscopy. The fusion process is critical for exchanging mitochondrial contents and maintaining integrity of the mitochondrial genome, whereas fission is vital for being able to distribute mitochondria to subcellular locations where atp is most needed or during mitosis, and. Introduction membranepotentialdependent dyes such as rhodamine 123, tetramethylrhodamine methyl ester tmrm, and tetramethylrhodamine ethyl ester tmre are useful as long as the mitochondrion maintains its negative membrane potential. Choosing proper fluorescent dyes, proteins, and imaging techniques. You will receive your score and answers at the end. Mitotracker probes and mitochondrial membrane potential. By co labeling the mitochondria with matrixtargeted pagfp and dsred2, the fusion and fission of mitochondria can be quantitatively recorded after irradiation at regions of interest in a chosen cell twig et al. Thus, this dye and tmrm were loaded and equilibrated prior to administration of toxicants section 2. Although they have distinct properties that allow the study of the functions, morphology and location of mitochondria, they share a. Ertracker dyes for livecell endoplasmic reticulum labeling.
Our laboratory has demonstrated the critical role of mitochondria in the progression of hyperglycemiamediated neuronal damage vincent and feldman 2004. Proteins in the outermembrane of the mitochondria which play an important role in fusion of mitochondria. Mitotracker labeling in primary neuronal and astrocytic. A high content imaging flow cytometry approach to study. Mitotracker deepred fm, and imaged using a threecolour wide. Co labeling with mitotracker orange or mitotracker green revealed almost complete overlap of the mitotracker dyes with the structures stained with r123 and tmre fig. Remove medium from the cells and add prewarmed medium with mitotracker most commonly used concentration is in the range of 300400 nmol. Apr 10, 2015 a flow cytometric analysis of mitochondrial membrane potential and size monitored by mitotracker staining on the indicated day top or differentiation of the b cells monitored by cd8 and igg1. Cytometric assessment of mitochondria using fluorescent probes. Sep 26, 2014 specific fluorescent mitochondrial dyes jc1, mitotracker red fm, and mitotracker red cmxros were studied on cultures of mesenchymal stromal stem cells from human adipose tissue.
Mitochondria in cells are seen as bright spots in the cytoplasm. This is an online quiz called mitochondria labeling. A variety of dyes are available for labeling mitochondria 28. Other quizzes cover topics on the scientific method, microbes, plants, invertebrates, vertebrates, and animal systems. Extracellular antiangiogenic proteins augment an endosomal. Note the loss of total mitochondria and the decrease in interconnectivity upon removal of 4ht.
Mitotracker green fm appears to localize to mitochondria regardless of mitochondrial membrane potential. Mitochondria structure thermo fisher scientific us. Several of the acridine dyes, first isolated in the nineteenth century, are useful as fluorescent probes in confocal microscopy. Additionally, we show that a biotinlabeled mitoblue derivative can be used as a handle for the delivery of streptavidintagged species to the. Visualizing mitochondrial lipids and fusion events in. Mitochondria are dynamic organelles that undergo constant remodeling through the regulation of two opposing processes, mitochondrial fission and fusion. Labeling mitochondria with mitotracker dyes request pdf. As per karen hales, a faculty of davidson collegenorth carolina, mitochondria fusion is a process where mitochondria constantly fuse merge and divide to form tubular networks. Full text pdf protocol labeling mitochondria with mitotracker dyes.
1069 1638 1118 389 78 991 1128 663 1569 1259 1358 661 560 688 360 1376 509 693 904 1306 864 524 1419 207 571 1267 946 277 677 1261 88