A Microplate-Based Bioluminescence Assay of Mitochondrial Calcium Uptake

Mitochondrial Ca²⁺ homeostasis is crucial for regulating vital functions such as respiration or apoptosis. Targeted aequorins are excellent probes to measure subcellular Ca²⁺. Ca²⁺ concentration in mitochondria ([Ca²⁺]_M) is low at rest (about 10^-7 M) and can increase to the micromolar or even approach the millimolar range, upon cell activation. Here we describe a new quantitative luminescent protocol to directly measure mitochondrial Ca²⁺ uptake, optimized for high throughput. The sensitivity of the method allows detection of changes in either the capacity or the affinity of mitochondrial Ca²⁺ transport.