Assaying Isolated Mitochondria in the Seahorse XF24: Learning More while Requiring Less.
Anne Murphy, PhD
Associate Adjunct Professor of Pharmacology
Department of Pharmacology
University of California at San Diego
Webinar Abstract:
The Seahorse XF24 Analyzer instrument was originally designed for use with intact cells attached to a tissue culture plate. However, as many researchers routinely utilize isolated mitochondria to answer fundamental questions about mitochondrial function, bioenergetics, and metabolism, we have developed a protocol that expands the application of the XF24 analyzer to include isolated mitochondria. We have successfully optimized conditions to assess rates of respiration and proton extrusion by isolated mitochondria attached to the bottom of the XF assay plate. This method has the distinct advantages of requiring extremely small quantities of material per well (5-10 mcg of mitochondrial protein) and allowing relatively high-throughput analysis of isolated mitochondrial samples. Sequential measurement of State II, State III, State IV, and uncoupler-stimulated respiration results in rates comparable to those generated using a conventional Clark-type electrode apparatus. However, acquisition of optimal rates requires assay conditions that are distinct from those of conventional polarography.
Join us for this 45 minute webinar in which the optimal conditions for the assay of isolated mitochondria will be described.
You Will Learn:
- How to assay ADP-stimulated, resting, and uncoupler stimulated respiration of isolated mitochondria using as little as 5 mcg of protein per well.
- How to develop and optimize appropriate assay conditions for isolated mitochondria in the XF24.
- How to trouble-shoot the quality of mitochondrial preparations and XF assay parameters to obtain successful and data-rich results.
Assay:
Mitochondrial Function: ETC Complexes Function/Couping
Substrate Utilization: Glutamate/Malate, Succinate
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