AAT Bioquest

Selective Probes for Studying Mitochondrial Functionality

It is indisputable that mitochondria are the primary producers of cellular energy via oxidative phosphorylation and lipid oxidation. In addition to their role as the cell's powerhouse, mitochondria have a key role in other vital cell physiological and pathological processes. For example, mitochondria play a role in regulating intrinsic pathways of apoptosis. They also contribute to buffering and shaping cytosolic calcium, and are potent producers of cellular reactive oxygen species (ROS), such as superoxide (•-O2−). Furthermore, mitochondrial dysfunction is a characteristic of many chronic diseases, including neurodegenerative and cardiovascular diseases, and chronic fatigue.

AAT Bioquest offers a broad selection of tools designed to investigate mitochondrial morphology and functionality, and ROS imaging. The following discussion highlights assay reagents and kits optimized for mitochondrial ROS imaging, as well as, exploring a convenient method for improving Rhod-2 sensitivity at detecting mitochondrial calcium. For a detailed overview of the various tools we provide to monitor mitochondrial morphology and functionality refer to the Table 1-2.


Mitochondria ROS Imaging

A byproduct of mitochondrial oxidative phosphorylation is ROS. These chemically reactive, oxygen-containing species are released into the cytoplasm to promote redox signaling. However, an improper balance of ROS production-detoxification leads to oxidative stress (ROS overproduction), which is characterized by the potentially damaging levels of intracellular ROS. Because ROS overproduction is detrimental to mitochondria it must be neutralized in order maintain normal cellular homeostasis. Failure to do so will result in damage to the organelle's lipids, proteins and DNA, and can induce cell damaging activities such as the induction of the mitochondrial permeability transition pore (mPTP). Opening of the mPTP channel is accompanied by an increase in the inner membrane permeability, the dissipation of the mitochondrial membrane potential, and a decrease in ATP production. The changes induced by the opening of mPTP can induce cell death.

Consequently, all of these different aspects of mitochondrial ROS have also been implicated in various pathologies including ischemia reperfusion injury, myocardial infarction, and chronic disorders (e.g., neurodegenerative disease, or type-2 diabetes). As such, the detection and quantification of mitochondrial ROS is important to understanding proper cellular redox regulation and the impact of its dysregulation on various cellular pathways and pathologies.

Superoxide Radical

Mitochondria are potent producers of cellular superoxide (•-O2−), a by-product of mitochondrial respiration (most notably by Complex I and Complex III). Superoxide is the predominant ROS in mitochondria. Superoxide, at low to moderate levels, is critical for the proper regulation of many essential cellular processes including gene expression and signal transduction. However, studies have shown mice deficient for mitochondrial superoxide dismutase (enzyme that neutralizes superoxide radicals) die due to neurodegeneration, cardiomyopathy and lactic acidosis (pathologies commonly correlated with elevated superoxide levels) (Muller et al. 2007). Therefore, investigating mitochondrial superoxide (exogenous or endogenous) levels are of great interest to study these medically relevant pathologies.

Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit

Fluorescence response of MitoROS 580
Fluorescence response of MitoROS™ 580 (10 µM, Cat# 16052) to different reactive oxygen species (ROS) and reactive nitrogen species (RNS). The fluorescence intensities were monitored at Ex/Em = 540/590 nm.
MitoROS™ 580 shares similar characteristics with dihydroethidium (a widely utilized superoxide indicator) and Invitrogen's MitoSOX™ Red. Therefore, experiments utilizing dihydroethidium or MitoSOX™ Red can be replaced with MitoROS™ 580 without any sacrifice in assay sensitivity. In fact, MitoROS™ 580 is less likely to be oxidized by other reactive oxygen species and reactive nitrogen species (Figure 1).MitoROS™ 520 and MitoROS™ 580 are two unique superoxide indicators and are the central components of our Cell Meter™ Fluorimetric Mitochondrial Superoxide activity assays. The cationic property of both probes facilitates their diffusion across the cytoplasmic and mitochondrial membranes as well as promotes their potential driven uptake within the mitochondria via the mitochondrial membrane potential. Similar to other commercially available •-O2- indicators, MitoROS™ 520 and MitoROS™ 580 probes are activated through oxidation by superoxide. Activated MitoROS™ 520 and MitoROS™ 580 probes then intercalate with DNA, which is essential for the generation of their strong fluorescence signals. Upon excitation, MitoROS™ 520 generates a green fluorescence while MitoROS™ 580 fluoresces red.

MitoROS™ 520 is unique in that it generates a strong green fluorescence when it is oxidized by superoxide and binds to DNA (Figure 2). Utilization of the green spectrum creates enough spectral separation from other red ROS indicators, such as MitoROS™ OH 580 (a hydroxyl radical indicator) making it suitable for the simultaneous monitoring of superoxide and hydroxyl radical levels. Or one can investigate mitochondrial calcium uptake and ROS production using MitoROS™ 520 and Rhod-2, a red calcium indicator.

Fluorescence images of superoxide measurement.

Fluorescence images of superoxide measurement in macrophage cells using Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit (Cat#16060). RAW 264.7 cells at 100,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom plate. AMA Treatment: Cells were treated with 5 µM Antimycin A (AMA) at 37 °C for 2 hours, then incubated with MitoROS™ 520 for 1 hour. Untreated Control: RAW 264.7 cells were incubated with MitoROS™ 520 at 37 °C for 1 hour without AMA treatment. The fluorescence signal was measured using fluorescence microscope with a FITC filter.

Hydroxyl Radical

The hydroxyl radical (•-OH) is another byproduct of oxidative metabolism and is the most reactive ROS. Hydroxyl radicals can produce DNA damage and cause significant subcellular damage to organelles. Therefore, sensitive and selective detection of intracellular •-OH is vital to understanding cellular redox and the impact of hydroxyl radicals on dysregulation in cells. While a variety of •-OH fluorogenic sensors are commercially available, many suffer from rapid photobleaching, short emission wavelengths, and non-selective reactions with other ROS species. All these factors have limited the applications in cells and tissues. To resolve these limitations, AAT Bioquest has dedicated extensive research and development in producing MitoROS™ OH580, a robust and sensitive •-OH fluorogenic sensor.

Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit

Fluorescence images of Hydroxyl radical measurement
Fluorescence images of Hydroxyl radical measurement in HeLa cells using Cell Meter™ Mitochondriol Hydroxyl Radical Detection Kit (Cat#16055). Control (Top): HeLa cells were kept in 1X HBSS buffer without treatment. Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17530). Fenton Reaction (Bottom): Cells were then treated with 10 µM CuCl2 and 100 µM H2O2 in 1X HBSS buffer at 37 °C for 1 hour. After washing 3 times with HHBS, HeLa cells were measured using a fluorescence microscope with a TRITC filter set (Red).
MitoROS™ OH580 is the main component of our Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit (Cat 16055). This novel fluorogenic indicator is cell-permeant and highly selective for hydroxyl radicals over other ROS, such as superoxide or hydrogen peroxide. MitoROS™ OH580's specific interaction with •-OH greatly minimizes non-specific background fluorescence and significantly enhances intracellular •-OH signals. Upon association with •-OH, MitoROS™ OH580 exhibits a strong red fluorescence. This fluorescence signal can be monitored by fluorescence microscopy or measured using a fluorescence microplate reader at Ex/Em = 540/590 nm. MitoROS™ OH580's utilization of the red spectrum makes multiplexing possible with mitochondrial morphological probes of green fluorescence.

Mitochondrial Calcium Imaging

Calcium uptake and efflux in mitochondria is important in regulating cytosolic calcium concentrations and metabolic processes. Mitochondrion possess the ability to rapidly take up calcium, primarily through the mitochondrial calcium uniporter (MCU) located on the inner mitochondrial membrane. Calcium influx into the mitochondrial matrix is dependent upon the organelles electrochemical gradient for calcium. This gradient is established and maintained by mitochondrial respiration, while the mitochondrial sodium-calcium antiporter is responsible for sustaining a low resting intramitochondrial calcium concentration.

Intramitochondrial calcium plays an intimate role in regulating mitochondrial metabolism by targeting three key rate limiting dehydrogenases of the citric acid cycle (CAC). Calcium directly binds to the enzyme complexes and activates α-ketoglutarate and isocitrate dehydrogenases. Pyruvate dehydrogenase activation is mediated through a calcium-dependent dephosphorylation step. Calcium activation of these dehydrogenase enzymes in mitochondria leads to an increase in NADH and the production of ATP. Disruption of mitochondrial calcium uptake alters the spatiotemporal properties of cytosolic calcium signaling and suppresses mitochondrial metabolism, which leads to a decrease in ATP production. This type of disruption of mitochondrial calcium uptake has been found to cause diseases such as chronic fatigue.

Rhod-2: Mitochondrial Calcium Indicator

Rhod-2, AM can be used to measure changes in mitochondrial calcium inside cells. Rhod-2, AM is a rhodamine-based calcium indicator that is conjugated with AM esters to become cationic.

This is advantageous for two reasons: 1) the cationic charge on Rhod-2, AM promotes the potential driven accumulation of Rhod-2 within mitochondria via mitochondria membrane potential 2) AM esters facilitate the diffusion of Rhod-2 across the cytoplasmic membrane as well as the mitochondrial membrane. Once inside the mitochondria, non-specific intracellular esterases remove the AM groups, which traps Rhod-2 inside the mitochondria. Rhod-2 exhibits a significant increase in fluorescence intensity upon association with calcium ions.

Rhod-2 has a fluorescence excitation and emission maxima at 549 nm and 578 nm, respectively. This characteristically long-wavelength makes it suitable for cellular and tissue imaging which have relatively high autofluorescence. This is an advantage for detecting calcium release generated by photoreceptors and photoactivatable chelators.

One of the disadvantages of Rhod-2 AM is its susceptibility to background interference. Non-specific intracellular esterases are not unique to mitochondria and are present throughout the cytosol. When Rhod-2 AM is loaded into cells, the indicators can be activated and retained within the cytosol before it can be sequestered within mitochondria. A solution for improving Rhod-2 sensitivity is to reduce it to dihydrorhod-2 AM prior to use.

Improving Rhod-2 Sensitivity: Method to Reduce Rhod-2 to Dihydrorhod-2

Reduction of Rhod-2 AM to dihydrorhod-2 AM enhances its mitochondrial localization and reduces background interference. This change improves sensitivity because it requires a two-part activation process. The non-fluorescent dihydrorhod-2 AM can only exhibit calcium-dependent fluorescence after oxidation and AM cleavage in the mitochondria. Residual dihydrorhod-2 retained in the cytosol, due to AM cleavage, cannot be taken up by the mitochondria for oxidation and will remain non-fluorescent even in the presence of cytosolic calcium. Reduction of Rhod-2 AM to dihydrorhod-2 AM can be readily achieved using the protocol below:
  1. Dissolve 50 µg of Rhod-2 AM in 100 µL of anhydrous dimethylsulfoxide (DMSO).
  2. Add a small excess (the smallest amount solid that can be practically transferred is sufficient enough) of solid sodium borohydride (NaBH4 ).
  3. Incubate for 10 minutes or until the reaction mixture appears colorless.
  4. Use the reaction solution in DMSO (about 0.4 mM dihydrorhod-2 AM) directly for cell loading according to usual AM ester loading protocols.

*Note: Dihydrorhod-2 AM will spontaneously and rapidly revert to its oxidized form, this is indicated by reappearance of color in your stock solution. So, experiments using dihydrorhod-2 AM should be carried out immediately after preparation.

Appendix: AAT Bioquest Mitochondrial Tools

The following tables provide a summary of the AAT Bioquest's mitochondrial tools and stains as well as their targets.

Table 1. AAT Bioquest tools to study mitochondrial morphology.
ProbeCatalog#Ex/Em (nm)FixableApplicationPrinciple
MitoLite™ Blue FX49022674350/490YesLive cellCationic fluorogenic probe sequestered by functioning mitochondria via the mitochondrial membrane potential gradient (ΔΨM). MitoLite™ FX dyes retain fluorescent staining pattern after fixation and permeabilization.
MitoLite™ Green EX 48822675498/520No
MitoLite™ Green FM22695491/513Yes
MitoLite™ Orange EX40522679399/550No
MitoLite™ Orange FX57022676545/575Yes
MitoLite™ Red FX60022677575/600Yes
MitoLite™ Deep Red FX66022678640/659Yes
MitoLite™ NIR Red FX69022690660/692Yes

Table 2. AAT Bioquest tools to study mitochondrial functionality
TargetToolCatalog#Ex/Em (nm)ApplicationPrinciple
Mitochondrial Calcium FluxRhod-2, AM





549/578Live CellsCationic calcium indicator. This unique property allows it to be sequestered in mitochondria via the ΔΨM. Cleavage by esterases activates rhod-2's calcium-dependent fluorescence. Upon excitation rhod-2 fluoresces red.
Mitochondrial Membrane Potential ProbesRhodamine 12322210507/529Live Cells

These cell-permeable, cationic probes localize in the mitochondria via the ΔΨM and are designed to quantify changes in ΔΨM. Mitochondria with decreased ΔΨM will fail to sequester probes. Upon excitation Rhodamine 123 will fluoresce green. TMRM and TMRE both will fluoresce red-orange.

JC-122200515/529Live Cells

JC-1 is a cationic probe used to determine ΔΨM. It exhibits potential-dependent uptake by mitochondria which is represented by a fluorescence emission shift from green to red. At low concentrations, JC-1 exists as a monomer and when excited it fluoresces green. At higher concentrations, the dye forms 'J-aggregates' which fluoresces red.





510/525Live Cells

JC-10™ is a superior alternative to JC-1. Its improved solubility and higher signal-to-noise ratio makes for a more convenient and robust assay. JC-10TM's potential-dependent spectroscopic properties are identical to JC-1.

Mitochondrial Oxidative PhosphorylationMitoROS™ 58016052510/580Live Cells

These probes passively permeate intact cells and sequester within the mitochondria. Probes are activated via oxidation by superoxide. Upon activation probes intercalate with DNA and fluoresce upon excitation. DNA binding is necessary for strong fluorescence signal.

MitoROS™ 52016060509/534Live Cells

Probe readily diffuses through intact cell membranes and selectively accumulates in mitochondria. It is oxidized by superoxide and when excited it generates a green fluorescence.

MitoROS™ OH58016055576/598Live Cells

Probe freely permeates lives cells where it selectively targets free hydroxyl radicals. Oxidation by hydroxyl radicals activates the probe and when excited it generates a red fluorescence.



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