Quantitation of mitochondrial alterations associated with apoptosis

Mitochondria undergo two major changes during early apoptosis. On the one hand, the outer mitochondrial membrane becomes permeable to proteins, resulting in the release of soluble intermembrane proteins (SIMPs) from the mitochondrion. On the other hand, the inner mitochondrial membrane transmembrane potential (ΔΨ_m) is reduced. These changes occur in most, if not all, models of cell death and can be taken advantage of to detect apoptosis at an early stage. Here, we delineate methods for the detection of alterations in the ΔΨ_m, based on the incubation of cells with cationic lipophilic fluorochromes, the uptake of which is driven by the ΔΨ_m. Certain ΔΨ_m-sensitive dyes can be combined with other fluorochromes to detect simultaneously cellular viability, plasma membrane exposure of phosphatidylserine residues, or the mitochondrial production of reactive oxygen species (ROS). In addition, we describe an immunofluorescence method for the detection of two functionally important proteins translocating from mitochondria, namely, the caspase co-activator cytochrome c and the caspase-independent death effector apoptosis inducing factor (AIF).