In Vitro Cell Death Determination for Drug Discovery: A Landscape Review of Real Issues

Méry, Benoite; Guy, Jean-Baptiste; Vallard, Alexis; Espenel, Sophie; Ardail, Dominique; Rodriguez-Lafrasse, Claire; Rancoule, Chloé; Magné, Nicolas

lei. Another method widely used to study and measure DNA fragmentation is the terminal dUTP nick end labeling technique based on the formation of endonuclease-generated DNA breaks that are enzymatically labeled by terminal transferase with uridine triphosphate derivatives coupled with fluorochromes; secondarily, an immunoperoxidase reaction allows the detection. DNA fragmentation is then evaluated quantitatively by fluorescence microscopy or by flow cytometry. One has to be vigilant about the risk of false-positives linked to necrotic cells, and this method should be paired with additional assays. 38, 39 Mitochondrial membrane depolarization (MMD) occurs at early apoptotic stages and before the primary morphologic and biochemical alterations. Therefore, membrane potential monitoring can provide information about kinetics of apoptotic processes. A variety of potentiometric dyes, with both flow cytometry and fluorescence microscopy, can be used for the measurement of the mitochondrial membrane potential. However, the hypothesis of MMD as an early marker of mitochondrial alterations is largely controverted, and the decrease in the transmembrane potential might rather be considered as a marker of mitochondrial damage that may take Méry et al 5 place in late apoptosis and also during necrosis. 40 Beyond MMD, the MOMP process can also be investigated through the release of apoptogenic factors, including cytochrome c, Smac, or endoG, from the mitochondria to the cytosol. Finally, if apoptosis was initially characterized by specific morphologic features, we must keep in mind that microscopic analysis of cells is still a major way for studying apoptosis, for instance, through microscopic analysis of chromatin condensation after DNA staining.