Assessing Calcium-Stimulated Mitochondrial Bioenergetics Using the Seahorse XF96 Analyzer
The development of fluorescence-based oxygen sensors coupled with microplate-based assays for quantitative bioenergetics analyses enables screening multiple experimental conditions at once with small biological material and in a timely manner. In this chapter, we outline detailed protocols and practical tips to design and perform controlled measurements of (a) respiratory and glycolytic metabolism of intact cells, (b) substrate-dependent respiration in permeabilized cells and isolated mitochondria, and (c) calcium-dependent regulation of mitochondrial bioenergetics with Seahorse XF Flux Analyzers.
Key wordsMitochondria Bioenergetics Respiration Oxygen consumption Intact cells Permeabilized cells Seahorse XF analyzer Calcium
We thank Dr. Daniela Arduino and Valerie Goh for critical reading of the manuscript. This work was supported by the German Research Foundation (DFG) under the Emmy Noether Programme (PE 2053/1-1) and the Bert L & N Kuggie Vallee Foundation (to F.P. and J.W.).
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