Apoptosis mitochondrial assays

A promising tool for toxicity evaluation by rapid high throughput screening is the mitochondrial ADP/ATP ratio assay, which is sensitive and can detect several highly energy dependent processes, such as apoptosis, necrosis, growth arrest, and cell proliferation [68],... 

From the aforementioned description of mitochondrial physiology and the MPT, it is clear that pore opening and dissipation of the proton gradient will result in relative alkalinization of the inner mitochondrial space and acidification of the cytoplasm. Fluorescence-based assays to detect altered cellular pH and their use in apoptosis will be addressed below, as will alterations in intracellular calcium concentrations. Similiarly, although the mitochondrial electron-transport chain is an important source of reactive oxygen... 

As with flow cytometry, multiparameter apoptosis assays may also be performed by confocal laser scanning microscopy (CLSM). Using the approach similar to that detailed above for flow cytometry, we have examined NADPH content, mitochondrial membrane potential (CMX Rosamine fluorescence), and mitochondrial mass (Mitotracker Green), by CLSM. Figure 3 shows an example of a typical multiparameter assay performed by confocal microscopy. 

Enzyme-linked immunosorbent assays (ELISAs) have long been used to quantify cytokines and other proteins that are secreted from cells. ELISAs enable the user to assay multiple samples, to obtain reproducible quantitative results, and to design studies with quantifiable endpoints. Intracellular activities intimately involved in apoptosis, such as control of mitochondrial permeability to holocytochrome c and activation of a specific caspase, are quantified by the ELISAs described in this chapter. The cytochrome c ELISA is generally used to quantify the activities of the proteins belonging to the Bcl-2 family. The active caspase ELISA quantifies a specific active caspase among a background of latent caspase and other active caspases. 

As mentioned above, cytotoxicity evaluation is one of the applications on iPSC-derived disease models [104]. With the use of a high-content assay, a series of readouts, such as cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis, have been examined as a general and mechanism-specific hepatotoxicity. In this particular application, the cell model for the assay... 

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