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.
McConkey
David J
DJ
Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, USA.
Nutt
Leta
L
D000331:Aequorin / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019284:Thapsigargin / Q000378:metabolism
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D000331:Aequorin; D019284:Thapsigargin; D002118:Calcium