CELLULAR METABOLISM ASSAYS

Apoptosis assays

Apoptosis can be activated by stimuli coming within the cell, including cell stressors, such as hypoxia or lack of nutrients, and agents that cause damage of DNA or other cell structures. In vertebrates this pathway is initiated by the release of apoptosis mediators from the mitochondrial intermembrane space, when certain intracellular signals permeabilize the outer membrane of the mitochondria.

 

A major protein in this process is cytochrome c, component of the electron transport chain on the external face of the inner mitochondrial membrane. When released into the cytosol, cytochrome c binds to APAF-1, which oligomerizes to form a complex or apoptosome. Once formed this complex, comprising APAF-1–cytochrome c–procaspase 9, triggers the activation of procaspase 9 to caspase 9. This initiator protease starts the hydrolytic cascade that stimulates the effector caspases (3, 6, and 7).

 

Oxidative stress has been shown to participate in a wide range of diseases including atherosclerosis, chronic obstructive pulmonary disease (COPD), Alzheimer disease and cancer, which has revealed the multiple mechanisms by which oxidants contribute to cellular damage.

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Cell Viability, Cytotoxicity, MMP and Apoptosis Assays
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Cytotoxicity and Cell Viability assays

Cell viability and cytotoxicity assays are used for drug and cosmetic ingredient screening, and cytotoxicity tests of chemicals. In vitro cytotoxicity testing provides a crucial means of ranking compounds for consideration in drug discovery. The choice of using a particular viability or cytotoxicity assay technology may be influenced by specific research goals.

In vitro methods have become a cornerstone of drug discovery and are widely applied both for screening and mechanistic studies. Whether researching cytotoxic, deleterious (toxic), or protective effects, the determination of concentrations that are cytotoxic to the model should be the primary step of in vitro testing.

Depending on the research scopes and on the further aims that are expected to be met, cytotoxicity may or may not be an endpoint on its own.

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Mitochondrial Membrane Potential (MMP) assays

Mitochondria, commonly referred to as power houses of the cell, play a vital role in cellular physiology. The majority of the cellular energy (ATP) in eukaryotic cells is generated in the mitochondria through oxidative phosphorylation, during which electrons are transferred from electron donors to electron acceptors such as oxygen. The mitochondrial electron transport chain creates an electrochemical gradient through a series of redox reactions. This electrochemical gradient drives the synthesis of ATP and generates the mitochondrial membrane potential (MMP), which is a key parameter for evaluating mitochondrial function.

Mitochondrial dysfunctions have been associated with various disorders such as cancer, cardiovascular diseases, diabetes, and neurodegenerative diseases. The toxicity of xenobiotic compounds can have either a direct or a secondary effect on mitochondrial function. Many of these compounds reduce MMP by perturbing a variety of macromolecules in the mitochondria, and therefore affecting different mitochondrial functions. A decrease in the MMP may also be linked to apoptosis. Thus these organelles are an ideal target for in vitro toxicity studies.

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Cell Metabolism, Oxidative Stress and Enzyme Assays
Cellular Metabolism, Energy and Oxidative Stress Markers

Oxidative stress involves the chemistry of reactions of so- called reactive species derived from oxygen and nitrogen, particularly •OH, ONOO− and HOCl. The second mechanism of oxidative stress is aberrant redox signalling. Oxidants, particularly H2O2 generated by cells upon physiological stimulation, can act as second messengers.

To defend against oxidative injury, organisms have evolved defences primarily dependent upon antioxidant enzymes.

The energy released from the breakdown of the chemical bonds within nutrients can be stored either through the reduction of electron carriers or in the bonds of adenosine triphosphate (ATP).

In living systems, a small class of compounds functions as mobile electron carriers, molecules that bind to and shuttle high-energy electrons between compounds in pathways. The principal electron carriers are NAD, NADP and FAD. These compounds can be easily reduced or oxidized. NAD+/NADH is the most common mobile electron carrier used in catabolism. NADP+, the oxidized form of an NAD+ variant that contains an extra phosphate group, is another important electron carrier; it forms NADPH when reduced. NAD+/NADH are extensively used in energy extraction from sugars during catabolism in chemoheterotrophs, whereas NADP+/NADPH plays an important role in anabolic reactions and photosynthesis.

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in Roles of Vascular Oxidative Stress and Nitric Oxide in the Pathogenesis of Atherosclerosis, Ulrich Förstermann, Circulation Research, Volume: 120, Issue: 4, Pages: 713-735, DOI: (10.1161/CIRCRESAHA.116.309326)

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Fluorometry Enzymatic assays
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