Measurement of Changes in Intracellular Calcium During Apoptosis

The role of Ca²⁺ changes in the commitment to apoptosis has been appreciated for more than two decades. However, early work focused on increases in cytosolic Ca²⁺ levels that may not be associated with most examples of programmed cell death. Rather, recent studies indicate that release of Ca²⁺ from the endoplasmic reticulum (ER) and subsequent mitochondrial Ca²⁺ uptake plays a more important role by regulating release of cytochrome c from mitochondria. These apoptosis-associated Ca²⁺ fluxes are regulated by members of the BCL-2 family of proteins and may therefore be critical targets of their evolutionarily conserved actions. Therefore, the availability of reliable techniques for measuring organelle-associated Ca²⁺ fluxes is critical to ongoing research in the field, yet these techniques present unique challenges not associated with the more routine measurements of cytosolic Ca²⁺ levels. In this chapter, we provide detailed methods for measuring cytosolic, ER, and mitochondrial Ca²⁺ levels in whole using commercially available fluorescent dyes, identifying key potential pitfalls and alternative strategies.