In Vivo and In Vitro Determination of Cell Death Markers in Neurons

Mitochondria are key regulators of cellular death. The mitochondrial membranes contain essential enzyme complexes for maintaining metabolic homeostasis and meeting the energy requirements of the cell (Tait and Green, Nat Rev Mol Cell Biol 11:621–632, 2010 and Galluzzi et al., Apoptosis 12:803–813, 2007). Thus, any perturbation of outer or inner mitochondrial membranes can lead to disruptions in the normal fluxes of key ions and metabolic proteins (i.e., ADP/ATP exchange), leading to eventual cellular death. In addition to maintaining cellular viability, mitochondria play a critical role in the initiation of programmed cell death. As initiators of the cell death process, key mitochondrial proteins [Cytochrome C (Cyt C) one of the most well-studied among them] are released from the intermembrane space during early cell death events eventually leading to caspase activation. Release of Cyt C is a crucial step during cellular death (Tait and Green, Nat Rev Mol Cell Biol 11:621–632, 2010). Therefore, the measurement of Cyt C release can give vital information about cell death signaling. Immunolabeling against Cyt C can give an easy readout of mitochondrial integrity as well, allowing for simultaneous identification of mitochondrial viability (and/or damage) and initiation of intracellular death processes. In this chapter, we use Cyt C as a dual marker of mitochondrial integrity and cell death and review several protocols to measure Cyt C localization into intact mitochondria and its release into the cytosol. The goal is to offer an array of assays that, combined, provide both qualitative and quantitative analysis of the relationship between mitochondrial viability and activation of an intracellular cell death process. Immunofluorescence, Western blot, and ELISA measurements of Cyt C as are discussed in detail.