High Throughput Microplate Respiratory Measurements Using Minimal Quantities Of Isolated Mitochondria

Abstract

Recently developed technologies have enabled multi-well measurement of O2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1–10 µg of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples.

Mitochondria are required for pro‐ageing features of the senescent phenotype

“…The resulting pellet was resuspended in a small volume of STE buffer for functional experiments. Mitochondrial oxygen consumption rates were measured using the Seahorse XF analyzer based on the method described in Rogers et al (2011), with adjustment explained in the Comments to the articles published in PLoS online (A source of data variation in mitochondrial respiration measurements). The mitochondria were energized with 5 mM pyruvate and 5 mM malate, and State III respiration was obtained by adding 4 mM ADP.…”

Glutaredoxin-2 Is Required to Control Proton Leak through Uncoupling Protein-3

“…Prior to HPLC, serum was diluted 10-fold. Mitochondrial Isolation-Skeletal muscle and liver mitochondria were isolated as described previously (11,35). All buffers were supplemented with 10 mM pyruvate and 2 mM malate or 10 mM succinate and filtered prior to use.…”

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

“…In addition, RCR can also be used as an indicator of coupled mitochondria. Therefore, the OCR and mitochondrial states within a typical range from the pyruvate/malate assay 8 , and the high RCR values obtained from a variety of respirometric assays indicates that the mitochondria derived from this protocol are tightly coupled and highly functional.…”

“…Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several µg per well 8 . Therefore, we present a modification of previously published methods 7 to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays.…”