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.
Highlights
Enhanced appreciation of the role of altered mitochondrial function in metabolic and cardiovascular disease, tumorigenesis, aging and degenerative diseases, and cell signaling has stimulated the development of a variety of new approaches for the assessment of mitochondrial function [1,2,3,4]
The O2 concentration values were reviewed (Fig. 2B). These data illustrate the result of overloading the wells, and show that with mouse liver mitochondrial samples of 10 mg or greater per well, a) O2 can be completely depleted from the microchamber (0.0 O2 tension), and b) the system does not have an adequate time to recover to normoxia before the measurement cycle
It is suggested that basal respiratory rates be kept between 100– 200 pmol/min/well to afford the best signal-to-noise ratio and dynamic range for the assay, and we find that, depending on tissue and species used, 1–10 mg of isolated mitochondria is optimal for the assay
Summary
Enhanced appreciation of the role of altered mitochondrial function in metabolic and cardiovascular disease, tumorigenesis, aging and degenerative diseases, and cell signaling has stimulated the development of a variety of new approaches for the assessment of mitochondrial function [1,2,3,4]. A change in intact cell respiration may owe to multiple potential alterations that cannot be distinguished without further experimentation, including the rate of ATP utilization, and the transport, storage and mobilization of added and endogenous substrates. As a result, it is often desirable and most informative to collect respiratory data with isolated mitochondria and be able to control the availability of substrates and ADP. We focused our efforts on developing an assay using isolated mitochondria in the XF24 analyzer, and have successfully devised a protocol that allows measurement of mitochondrial O2 consumption with as little as 1 mg of mitochondrial protein per well in a multi-well format, facilitating the quantity of information and minimizing the time it takes to gather respiratory data from small tissue samples
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