In spite of the critical roles that mitochondria play in all cells and the many ways they can be adversely affected by chemical compounds, mitochondrial toxicity might be crucial to identify during drug testing. Mitochondria can be affected by drug treatment, resulting in cardio- and hepatotoxic side effects that can lead to drug withdrawal from the market. In fact, the FDA suggests that, for example, all antiviral drugs should be tested for impact on mitochondrial function. A number of different methods can be used to monitor and evaluate changes in the morphology and function of the mitochondria.
ATP depletion assay
Mitochondria produce most of the cellular energy in the form of adenosine triphosphate (ATP) via oxidative phosphorylation. Many cell lines are metabolically adapted to growth in high glucose media and derive most of their energy from glycolysis rather than mitochondrial oxidative phosphorylation which reduces the susceptibility of the cells to mitochondrial toxicants. Using galactose as an energy source cells are forced to rely on mitochondrial oxidative phosphorylation for ATP production. To detect mitochondrial impairment we compare the toxic effects of different drugs on mitochondrial ATP production capability in the glucose and galactose media. For the measurement of ATP level we use assays based on luciferase activity and luminescence readout. In addition to the standard endpoint measurement of ATP in lysed cells, we are able to perform kinetic measurements in living cells.
| CellTitier-Glo™ | RealTime-Glo™ | |
| Working principle | Highly sensitive firefly luciferase substrate that reacts with ATP to generate a luminescent signal. | Assay measures extracellular ATP released by stressed cells. Assay reagent is added directly to cells and allows real-time monitoring. |
| Detection method | Luminescence | Luminescence |
| Platform | Plate reader | Plate reader |
| Sensitivity | 4/4 | 4/4 |
| Throughput | High | High |
| Multiplexing | Yes | Yes |
| Measurement type | Endpoint | Real-time |
| Model system | Adherent and suspension 2D cell cultures, 3D cell models | |
Example output:
Measurement of mitochondrial membrane potential
The electrochemical gradient across the inner mitochondrial membrane (mitochondrial membrane potential) is the driving force behind oxidative phosphorylation and its maintenance is critical for normal cellular function. Mitochondrial membrane potential as an integral component of cellular energy homeostasis is therefore a reasonable in vitro indicator of chemically induced cytotoxicity. We use a number of fluorescent dyes to accurately measure mitochondrial membrane potential. Tetramethylrhodamine methyl ester (TMRM), rhodamine123 or MitoTrackers passively diffuse across the plasma membrane and accumulate in active mitochondria generating single-component fluorescence signals. In contrast to these monochromatic probes, the ratiometric dyes like JC-1 and its derivatives (eg. Mito-ID) exhibit potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~525 nm) to red (~590 nm) with increasing concentration (i.e., aggregation). The ratio of green to red fluorescence depends only on the mitochondrial membrane potential and not on other factors such as mitochondrial size, shape, and density.
| JC-1 | MITO-ID® | TMRM, rhodamine123, MitoTracker™ | |
| Working principle | Assay utilizes cationic dye which enters mitochondria in potential-dependent manner. At high concentrations it aggregates and shifts its fluorescent emission spectra. | Similar to JC-1, the assay utilizes a cationic dye that enters mitochondria in a potential-dependent manner, where it aggregates, resulting in a shift in the fluorescent emission spectrum. | These fluorescent dyes enter active mitochondria allowing study of mitochondria function and dynamics. |
| Detection method | Fluorescence (ratiometric) | Fluorescence (ratiometric) | Fluorescence |
| Platform | Flow cytometer / Fluorescence microscope | Plate reader / Fluorescence microscope / Flow cytometer | Plate reader / Fluorescence microscope / Flow cytometer |
| Sensitivity | 3/4 | 4/4 | 4/4 |
| Throughput | Medium | High | High |
| Multiplexing | Yes | Yes | Yes |
| Measurement type | Real-time / Endpoint | Real-time / Endpoint | Real-time / Endpoint |
| Model system | Adherent and suspension 2D cell cultures | ||
Analysis of mitochondrial mass and morphology
Mitochondrial mass (amount of mitochondria per cell) can be the measure of mitochondrial toxicity. An increase in mitochondrial mass is thought to occur as a consequence of an adaptive response by the cell to increase energy production on the other hand mitochondrial damage can also manifest itself as a decrease in mitochondrial mass in the cell. Changes in the mitochondrial mass are often preceded by mitochondrial morphology transitions regulated by dynamic membrane fusion and fission processes. Both mitochondrial mass and morphology changes can be monitored in single cells with the use of fluorescent probes targeting mitochondria regardless of mitochondrial membrane potential.
| MitoTracker™ Green FM | 10-N-nonyl-acridine orange (NAO) | |
| Working principle | Mildly thiol-reactive chloromethyl probe which accumulates in active mitochondria. | Fluorescent probe which enters active mitochondria and is sensitive for depolirazation enabling real-time observations. |
| Detection method | Fluorescence | Fluorescence |
| Platform | Flow Cytometer / Fluorescence microscope / Plate reader | Flow Cytometer / Fluorescence microscope |
| Sensitivity | 4/4 | 4/4 |
| Throughput | High | High |
| Multiplexing | Yes | Yes |
| Measurement type | Endpoint | Real-time / Endpoint |
| Model system | Adherent and suspension 2D cell cultures | |
Oxidative stress
Mitochondria are considered a primary intracellular site of reactive oxygen species (ROS) generation. ROS are intimately involved in redox signaling and have a role in a number of cellular processes, but in some situations can also lead to oxidative damage. Oxidative stress occurs when there is an increased production of reactive oxygen species (ROS) or a decrease in the effectiveness of cellular antioxidant defenses. Assays to determine oxidative stress may measure levels of toxic reactive oxygen species or levels of cellular antioxidants (e.g. glutathione). We analyze the oxidative stress by measuring the generation of ROS in mitochondria as well as in the whole cell with the use of different fluorescent or luminescent probes. The level of oxidised form of glutathione (GSSG) or the ratio of reduced and oxidized forms (GSH/GSSG) is also an excellent indicator of oxidative stress in cells.
| MitoSOX™, H2DCF-DA | ROS-Glo™ | GSH/GSSG-Glo™ | |
| Working principle | Both dyes are susceptible to oxidation. MitoSOX™ is specific for mitochondrial superoxide, while H2DCF-DA offers a broader assessment of intracellular redox status. | The assay involves the conversion of a luminogenic substrate by H2O2, resulting in the generation of luminescence. | The assay involves the specific and sequential enzymatic reactions that result in the generation of luminescence, providing separate measurements for reduced (GSH) and oxidized glutathione (GSSG). |
| Detection method | Fluorescence | Luminescence | Luminescence |
| Platform | Flow cytometer, Fluorescence microscope, Plate reader | Plate reader | Plate reader |
| Sensitivity | 3/4 | 4/4 | 4/4 |
| Throughput | High | High | High |
| Multiplexing | Yes | No | No |
| Measurement type | Real-time / Endpoint | Endpoint | Endpoint |
| Model system | Adherent and suspension 2D cell cultures | ||
Activity of OXPHOS complexes
The electron transport chain (ETC) couples electron transfer between donors and acceptors with proton transport across the inner mitochondrial membrane. The resulting electrochemical proton gradient is used to generate chemical energy in the form of adenosine triphosphate (ATP). Proton transfer is based on the activity of complex I–V proteins in the ETC. The respiratory chain complexes are important for drug discovery in that they can be involved in toxic effects or might be of interest as drug targets themselves. We measure the inhibition of each complex activity in the presence of test compounds using MitoTox colorimetric activity assays.
| MitoTox™ OXPHOS activity assay | |
| Working principle | Series of assays designed for assessing the activity of different components of the oxidative phosphorylation (OXPHOS) pathway within mitochondria. Each assay targets a specific complex or enzyme, measuring its activity through enzymatic reactions or specific interactions. |
| Detection method | Colorimetric |
| Platform | Plate reader |
| Sensitivity | ++ |
| Throughput | Low |
| Multiplexing | No |
| Measurement type | Endpoint |
| Model system | Adherent and suspension 2D cell cultures, isolated mitochondria |
Example output:
