Functional assays

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:

The permeability of compounds across cell membranes (e.g., intestinal epithelium) is a critical characteristic that determines the rate and extent of absorption and ultimately affects the bioavailability of a drug candidate. We provide bidirectional permeability assays in human Caco-2 and MDCK cells.

Parallel Artificial Membrane Permeability Assay (PAMPA)

Drugs often need to cross cell membranes in order to reach their target of action and this makes a compound’s ability to passively cross these membranes an important characteristic to evaluate. The Parallel Artificial Membrane Permeability Assay (PAMPA) is used as an in vitro model of passive, transcellular permeation. The assay allows for measuring the gastrointestinal permeability of oral therapies, blood-brain barrier permeability, and dermal/transdermal penetration potential.

	Pion PAMPA
Detection method	Absorbance
Platform	Plate reader
Throughput	High
Multiplexing	No
Model system	Cell-free

Caco-2/MDCK permeability assay

The Caco-2 permeability assay utilizes Caco-2 cell line derived from human colon carcinoma, which has many characteristics that resemble intestinal epithelial cells. This assay offers a measure of the permeability across the intestinal barrier in both directions: the compound is added to the apical or basolateral compartment and efflux across the monolayer of cells is monitored. The amount of compound that has permeated across the cells is measured by LC-MS/MS.

The Madin-Darby Canine Kidney (MDCK) cell is an epithelial cell line derived from the canine kidney. The expression of transporter proteins and metabolic activity are low for MDCK cells but compared to Caco-2 cells, they proliferate and differentiate more quickly. Hence, it becomes an attractive alternative assay compared to Caco-2 permeability assay to assess the human intestine barrier.

	CacoReady	PreadyPort
Detection method	mass spectrometry	mass spectrometry
Throughput	High	High
Multiplexing	No	No
Measurement type	Endpoint	Endpoint
Model system	Caco-2 cell line	MDCKII cell line

Metastasis is the cumulative result of multiple changes in tumor cells and their microenvironment that enables cellular migration and invasion into healthy host tissue. Cell migration assays enable quantitative characterisation of cells movements, adhesion, and invasion and how these are influenced by pharmacological agents. We use different techniques to study these phenomena, from endpoint assays to time-lapse microscopy approaches and complex analysis for the downstream interpretation of the cell migratory behavior.

Wound healing assay

The wound-healing assay is a simple and inexpensive method to study directional cell migration in vitro. The basic step involves creating a “wound” in a cell monolayer manually or utilizing special microplates that provide a uniform and reproducible cell-free zone (i.e. Oris^™ Pro). Cells are then imaged at the beginning and at regular intervals during cell migration. It is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration. This assay is convenient and versatile and can be applied for a high throughput screen platform.

	Wound healing assay, Oris™ Pro
Detection method	Brightfield, Fluorescence
Platform	Microscope
Throughput	Medium
Multiplexing	No
Measurement type	Real-time
Model system	Adherent 2D cell cultures

Transwell cell migration/invasion assay

The most widely accepted cell migration technique is the Boyden Chamber assay. The classic transwell migration assay system uses a hollow plastic chamber, sealed at one end with a porous membrane. This chamber is suspended over a larger well that contains medium and/or chemoattractants. Cells are placed inside the chamber and allowed to migrate through the pores, to the other side of the membrane. To analyze cell invasion, the transwell insert membrane is coated with basement membrane ECM protein or a layer of cells such as endothelial cells. Migratory cells are then stained and directly counted in the microscope or some other indirect readout methods are used (e.g. metabolic activity assay).

	Boyden Chamber (transwell), ECMatrix™
Detection method	Fluorescence, Absorbance, Luminescence
Platform	Plate reader, microscope
Throughput	Medium
Multiplexing	Yes
Measurement type	Endpoint
Model system	Adherent and suspension 2D cell cultures

Microfluidic migration device chemotaxis assay

The alternative for the transwell system are microfluidic migration devices which promote a stable diffusion-generated concentration gradient. Slides are made from plastic with high optical qualities similar to those of glass allowing for life-microscopy assays. At specific time intervals, images of the observation area can be acquired, allowing real-time monitoring and quantitative measurements of cell migration.

	µ-Slide Chemotaxis, Millicell® µ-Migration Assay
Detection method	Fluorescence, Absorbance, Luminescence, Brightfield
Platform	Microscope, Plate reader
Throughput	Low
Multiplexing	No
Measurement type	Real-time
Model system	Adherent and suspension 2D cell cultures