Mitochondrial Toxicity Assay

Overview of Healthy Mitochondria

Mitochondria are the powerhouses of our cells. It is essential for energy generation, cellular metabolism, signaling, and cell death. Healthy mitochondria generally exhibit the following characteristics:

Inner Membrane Structural Integrity

Healthy mitochondria contain undamaged inner and outer membranes. Many folded structures known as mitochondrial cristae form on the interior of the inner membrane, where cellular respiration and energy production occur.

Respiratory Chain Function is Intact

Healthy mitochondria have a functioning respiratory chain, which includes the electron transport chain and the oxidative phosphorylation process. This permits mitochondria to convert dietary energy into ATP efficiently.

Oxidative Phosphorylation Activity

The primary energy production pathway in mitochondria is oxidative phosphorylation, performed by enzymes such as oxidative phosphatase. Healthy mitochondria have high oxidative phosphorylation activity, allowing cells to receive sufficient energy.

Adaptability to Anaerobic Conditions

When there is insufficient oxygen supply, healthy mitochondria can create ATP via processes such as anaerobic glycolysis to meet the cell's energy requirements.

Maintaining Membrane Potential

Healthy mitochondria can maintain an appropriate membrane potential, which is required for inner membrane permeability, cellular ion balance, and other vital cellular activities.

Self-Repair Mechanism

Healthy mitochondria can repair themselves and eliminate defective components. This helps to maintain overall mitochondrial function.

No Excessive Generation of Oxygen-Free Radicals

Under normal physiological settings, healthy mitochondria can efficiently restrict the formation of oxygen free radicals and thereby reduce cellular oxidative stress.

Fig.1 Healthy and damaged mitochondria.¹

When mitochondrial activity is compromised, a wide range of disorders can arise. Fig. 2 shows that mitochondria play a role in the etiology of human diseases and aging. Therefore, mitochondrial function testing is crucial for better understanding illness causation, diagnosis, and treatment.

Fig.2 Mitochondria are involved in the pathogenesis of human diseases, and aging.²

Mitochondrial Toxicity Assay

Creative Biolabs offers mitochondrial extraction and a variety of mitochondrial function testing services, including Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) using the Seahorse Energy Metabolism Analyzer, and fluorescence-based assays for membrane potential, superoxide, Reactive Oxygen Species (ROS), Ca²⁺, and Mitochondrial membrane permeability transition pores (MPTPs). OCR is an important indication of mitochondrial function. ECAR is a measure of lactic acid levels produced when glucose is converted to lactate during glycolysis. Superoxide is utilized to detect mitochondrial superoxide production, oxidative stress, and oxygen radical creation in living cells. ROS is used to measure intracellular ROS levels, which reflect oxidative stress levels and connect with cellular stress and disease development. Increased ROS indicates that mitochondrial dysfunction may lead to oxidative stress. Membrane potential is utilized to detect apoptosis at an early stage. Ca²⁺ is utilized to explore dynamic fluctuations in intracellular calcium ions. MPTPs are nonspecific channels in the inner and outer mitochondrial membranes that appear to have a role in the release of mitochondrial components after cell death.

• Mitochondrial Extraction

High-quality mitochondria are the primary condition for research on apoptosis, signal transmission, metabolism, and proteomics. We can isolate intact and purified mitochondria from animal cells or tissues relying on differential centrifugation, the two-step centrifugation of whole cell extracts, first at low speed to remove intact cells, cell and tissue debris, and nuclei, and then at high speed to concentrate mitochondria and separate them from other organelles.

• OCR & ECAR

We use the Seahorse Energy Metabolism Analyzer to continually measure oxygen concentration and proton flux in the cell supernatant. These observations are translated into OCR and ECAR values, allowing direct assessment of mitochondrial respiration and glycolysis.

• Membrane Potential

We can use fluorescent probes like JC-1 to detect membrane potential. In healthy cells, the mitochondrial membrane potential level is high. After entering the cells, JC-1 is easily enriched in the mitochondria and becomes multimers, showing red fluorescence. In apoptotic or diseased cells, the mitochondrial membrane potential decreases, and JC-1 appears in monomer form and emits green fluorescence. Changes in the fluorescence signal of the JC-1 probe were used to detect changes in mitochondrial membrane potential in apoptotic cells. Fluorescence signals can be used for flow cytometry result analysis, fluorescence microscopy observation, and 96-well fluorescence microplate reading.

• Superoxide

We can use fluorescent probes that specifically target mitochondria to selectively detect superoxide within mitochondria. The probes can penetrate living cell membranes and selectively enter mitochondria. Once inside the mitochondria, the probes can be oxidized by superoxide to fluoresce.

• ROS

We can use fluorescent probes such as DCFH-DA to detect ROS. DCFH-DA exhibits little fluorescence and can easily pass through the cell membrane. Once within the cell, it can be degraded by intracellular esterases to yield DCFH. DCFH cannot permeate cell membranes, allowing probes to be easily loaded into cells. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH, resulting in fluorescent DCF. Detecting DCF fluorescence can help measure the quantity of intracellular reactive oxygen species.

• Ca²⁺

We can use fluorescent probes like Fluo-3 and Fura-2 to detect mitochondrial calcium in living cells. As the calcium concentration in the mitochondria increases, so does the fluorescence of the calcium indicator.

• MPTPs

We can use acetoxymethyl ester (AM) of calcein and the calcein fluorescence quencher CoCl₂ to selectively label mitochondria. Calcein AM enters cells by passive diffusion and accumulates in organelles like mitochondria. Once inside the cell, calcein AM can be degraded by intracellular esterase to produce the extremely polar fluorescent dye calcein, which is unable to permeate the mitochondrial or cell membranes in substantial quantities in a short time. When the calcein fluorescence in the mitochondria is steady, add CoCl₂ to reduce the calcein fluorescence in the cytoplasm.

Creative Biolabs is committed to offering multiple mitochondrial function testing services. Our advantages are rich experience and mature technology. Please contact us for additional details.

References

1. Sorriento, Daniela, Eugenio Di Vaia, and Guido Iaccarino. "Physical exercise: a novel tool to protect mitochondrial health." Frontiers in physiology 12 (2021): 660068.
2. Javadov, Sabzali, Andrey V. Kozlov, and Amadou KS Camara. "Mitochondria in health and diseases." Cells 9.5 (2020): 1177.