AAT Bioquest

Fluorescent Tools for Studying Mitochondrial Morphology and Function

Mitochondria are the proverbial 'powerhouse' of eukaryotic cells producing a bulk of their energy supply in the form of adenosine triphosphate (ATP). As dynamic organelles, mitochondria can modify their architecture - sometimes taking up as much as 25% of a cell's volume - to accommodate for the cell's metabolic needs and other cellular processes, including signaling, cellular differentiation, cell cycle maintenance, cell growth, and apoptosis. In immunity, the importance of mitochondrial dynamics is apparent, as many pathogens have evolved mechanisms to modulate host cell mitochondrial remodeling and function to promote their survival. The retrograde signaling initiated by dysfunctional mitochondria can give rise to global changes in gene expression that affects cell morphology and function and initiates the propagation of disease processes. Alterations in mitochondrial morphology and function are good indicators of cell health, and multiplexing mitochondrial morphology reagents with probes that assess function can provide more in-depth information about mitochondrial health. AAT Bioquest has developed a wide range of mitochondrial stains and functional reagents to investigate mitochondria in live- and fixed-cell imaging applications. In this article, we highlight a few of our most-cited probes.


Mitochondrial Morphology Tools for Live-Cell Imaging

Co-localization using MitoLite™ Green FM. Live HeLa cells were incubated with the cell-permeant MitoLite™ Green FM (green) and the red-fluorescent lysosomal stain LysoBrite™ Red (red). Nuclei were counterstained with blue-fluorescent Nuclear Violet™ LCS1
Mitochondria morphology is complex, dynamic, and highly varied. Through tightly regulated fission, fusion, and mitophagy events, mitochondria can appear in various forms within a cell. From fragmented morphologies of small spheres and short to elongated tubules to a reticulated morphology in which the mitochondrion is a single network of many-branched structures. The number of mitochondria present and their location in a cell can also vary and depends on several variables, including cell or tissue type, developmental stage, metabolic requirement, and overall cell health. For instance, liver hepatocytes, which are involved in metabolism, detoxification, and protein synthesis, have about 1000-2000 mitochondria per cell. Because alterations in mitochondrial function result in dramatic modifications to its structure, staining mitochondria using fluorescent dyes such as MitoLite™ dyes and visualizing their morphology through a microscope can provide significant information about their overall biology, localization, and functional state (Table 1). Figure 1 shows HeLa cells with both spheroid-shaped mitochondria and mitochondria with normal reticulated morphology.

Table 1. AAT Bioquest tools for studying mitochondrial morphology.
TargetToolEx/Em (nm)Cat No.Mechanism of action
MitochondriaMitoLite™ Green FM508/52822695Non-fixable dyes that are sequestered by functioning mitochondria due to the organelle's transmembrane potential. However, cells stained with these dyes will lose their fluorescent staining patterns if mitochondrial function is disrupted or if cells are subjected to fixation and permeabilization.
MitoLite™ Green EX488508/52822675
MitoLite™ Orange EX405425/52222679
MitoLite™ Blue FX490344/46922674Fixable dyes that are sequestered by functioning mitochondria. Cells stained with these dyes retain their fluorescent staining patterns even if mitochondrial function is disrupted or if cells are subjected to fixation and permeabilization. This property makes them useful morphology markers that, once bound, are independent of mitochondrial function.
MitoLite™ Orange FX570553/57622676
MitoLite™ Red FX600580/59822677
MitoLite™ Red CMXRos578/59822698
MitoLite™ Deep Red FX660640/65922678
MitoLite™ NIR FX690658/69122690
CytoFix™ Red Mitochondrial Stain510/61023200Selectively stains mitochondria independent of their membrane potential and gives an index of mitochondrial mass based on staining intensity. After application, mitochondria can be imaged in live cells, or cells can be fixed and permeabilized for further study. This dye is suitable for long-term mitochondrial imaging and multiplex analysis with GFP expressed cells or other green-fluorescent probes.


Mitochondrial Functional Tools for Live-Cell Imaging

Mitochondrial hydroxyl radical detection
Mitochondrial hydroxyl radical detection. RAW 264.7 macrophage cells labeled with Nuclear Green™ LCS1 (green) and hydroxyl radical sensor MitoROS™ OH580 (red) were treated with PMA (phorbol 12-myristate 13-acetate) in a growth medium at 37 °C for four hours to stimulate endogenous hydroxyl radical.
Mitochondrial dysfunction - characterized by an inadequate number of mitochondria, an inability to provide necessary substrates to mitochondria, or the loss in efficiency of their electron transport chain and reductions in ATP production - is a hallmark of cellular toxicity and is associated with many chronic diseases. These include neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, cardiovascular diseases, diabetes, metabolic disorders, autoimmune diseases, gastrointestinal disorders, fatiguing illnesses, musculoskeletal diseases, and chronic infections. AAT Bioquest provides a variety of functional tools for studying mitochondrial function from various perspectives, including probes for mitochondrial membrane potential, autophagy/mitophagy, oxidative phosphorylation, calcium flux, and cytosolic pH (Table 2).

Table 2. AAT Bioquest tools for studying mitochondrial function.
TargetToolEx/Em (nm)Cat No.Mechanism of action
Mitochondrial membrane potentialTMRM552/57422221TMRM and TMRE are membrane-permeant, cationic rhodamine dyes for live-cell dynamic studies of mitochondrial membrane potential. They selectively accumulate in active mitochondria with intact membrane potential and, upon loss of potential, leaks into the cytoplasm. TMRM and TMRE are multiplexable with dyes that emit violet, blue, green, and deep red fluorescence, such as nucleic acid stains Hoechst 33342 and DAPI, the lipid stain Nile Green™, and lysosomal stain LysoBrite™ Deep Red
JC-1515/53022200JC-1 is a membrane-permeant, cationic carbocyanine dye for live-cell dynamic studies of mitochondrial membrane potential. It exhibits potential-dependent accumulation in mitochondria as indicated by a shift in fluorescence emission from 525 nm to 590 nm. At low concentrations due to low mitochondrial membrane potential, JC-1 exists as a monomer that yields green fluorescence with an emission of 530 nm. At high concentrations due to high mitochondrial membrane potential, JC-1 forms dye aggregates that yield orange fluorescence with an emission of 590 nm.
JC™-10508/52422204JC-10™ is an improved version of JC-1 offering better water solubility. JC-10 exhibits the same potential-dependent accumulation in mitochondria and shift in fluorescence emission from 524 nm to 590 nm. It can be used to study live-cell dynamics of mitochondrial membrane potential in flow cytometry, fluorescence imaging, and microplate assays.
Mitochondrial calcium fluxRhod-2 AM553/57721060Rhod-2 AM is a membrane-permeant calcium indicator that has utility in measuring mitochondrial calcium flux because of its preferential accumulation in mitochondria. Because of the low solubility of AM esters, a mild surfactant such as Pluronic® F-127 is typically used to disperse the dye and facilitate cell loading.
Oxidative phosphorylationMitoROS™ 520488/53016060Mitochondria generate various reactive oxygen species (ROS), particularly superoxides. MitoROS™ 520 is a membrane-permeant superoxide sensor for measuring mitochondrial superoxide levels in live cells. Upon reacting with superoxide, MitoROS™ 520 emits bright green fluorescence at 537 nm.
MitoROS™ 580500/58216052MitoROS™ 580 is a red superoxide indicator that permeates live cells and selectively targets mitochondria. The rapid oxidation of MitoROS™ 580 by superoxides produces highly fluorescent products that emit bright red fluorescence at ~580 nm.
MitoROS™ OH580576/59816055MitoROS™ OH580 is a membrane-permeant hydroxyl radical sensor that selectively targets mitochondria in live cells. Upon reacting with hydroxyl radicals, MitoROS™ OH580 emits bright red fluorescence at 598 nm.
Mitophagy (mitochondrial autophagy)Mitophagy Red™540/59022998Mitophagy Red™ readily permeates live cells where it selectively targets mitochondria. During the induction of mitochondrial autophagy, Mitophagy Red™ translocates to lysosomal compartments, and when excited, emits bright red fluorescence at ~590 nm. This dye is suitable for multiplex analysis with green-fluorescent mitochondrial morphology probes, GFP expressed cells, or other green-fluorescent probes (Figure 3).


High-Content Mitochondrial Analysis: Multiplexing Mitochondrial Morphology and Functional Tools

Mitochondrial morphology during mitophagy
Mitochondrial morphology during mitophagy. HeLa cells labeled with MitoLite™ Green FM (green) and autophagy sensor Mitophagy Red™ (red) were treated with CCCP to depolarize mitochondria. Loss of mitochondrial membrane potential triggers the targeted clearance of damaged mitochondria via mitophagy, as reflected through the colocalization of both dyes.
Multiplexing mitochondrial functional tools with morphology probes can significantly improve the study of mitochondria. For example, MitoLite™ Green FM can be combined with the mitophagy-sensitive dye Mitophagy Red™ to monitor the mitochondria-lysosome interactions vital for maintaining homeostasis in eukaryotic cells. These include mitochondria-lysosome fusion, a process that selectively removes redundant or damaged mitochondria (Figure 3), or mitochondria-lysosome contact (MLC). MitoLite™ dyes can be combined with potential-dependent probes such as TMRE or TMRM to monitor mitochondrial structural integrity while also assessing mitochondrial membrane potential, as well as with the mitochondrial calcium indicator Rhod-2 am or with mitochondrial ROS sensors.


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