1Buck Institute for Age Research, Novato, CA, 2Department of Pathology, Center for Free Radical Biology, University of Alabama at Birmingham - UAB, 3Seahorse Bioscience, North Billerica, MA
Nicholls, D. G., Darley-Usmar, V. M., Wu, M., Jensen, P. B., Rogers, G. W., Ferrick, D. A. Bioenergetic Profile Experiment using C2C12 Myoblast Cells. J. Vis. Exp. (46), e2511, doi:10.3791/2511 (2010).
The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates.
1. Cells Injection
The cells are metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell. One group will serve as the control, with running media added as control "compounds".
2. Reagents and Materials
3. Growth Medium
4. Seeding Protocol
5. Preparation of Assay Template
6. Compound Preparation
7. Media Change and Cell Preparation
8. Loading Sensor Cartridge
9. Protocol Commands
Table 1. Protocol commands
This assay is derived from the classic experiment to probe mitochondrial function and serves as a framework with which to build more complex experiments aimed at understanding various changes in cell metabolism, mitochondrial function, and overall bioenergetics.
All compounds used in this experiment should be optimized for the concentration that provides the maximal effect. That is, one must perform separate titration experiments to ascertain these values. This is especially important with FCCP, as the titration curve tends to be quite sharp, and too much FCCP can actually diminish responses in OCR. Typical ranges (final concentrations) to test would be:
Note that the responses to each compound above (especially FCCP) will be influenced by the assay media composition (base type, [glucose], [pyruvate], presence/absence of BSA, etc). Further, if the XF assay media composition is changed, optimization will need to be re-performed. The presence and concentration of pyruvate is especially important in obtaining the maximal respiratory capacity due to FCCP. Seahorse Bioscience has observed in a number of cells lines that omission of pyruvate abrogates the ability of cells to respond maximally (above baseline) to FCCP. Typically, concentrations of 1-10 mM pyruvate should be tested to understand the optimal concentration of pyruvate to obtain maximal respiration. Note that [pyruvate] AND [glucose] may need to be "cross-titrated" to obtain the optimal media conditions for the experiment.
Typical results of this experiment are presented below in a graph showing OCR vs. time and another showing ECAR vs. time:
Figure 2. OCR vs. Time
Figure 3. ECAR vs. Time
Here we observed the expected responses in OCR and ECAR as the cells are treated with each successive compound. For oligomycin, OCR decreases as a result of blocking ATP synthesis at mitochondrial Complex V. Since the cells are unable to synthesize ATP via OXPHOS, they revert to glycolysis to meet their demand for ATP, thus we observe an increase in ECAR. As shown previously, FCCP acts as an uncoupling agent. Since the cells must now overcome the proton leak across the inner mitochondrial membrane, OCR increases significantly as more O2 is consumed to pump the excess protons back across the mitochondrial membrane. Finally, rotenone inhibits mitochondrial Complex I and Complex III, respectively, which causes the flow of electrons to cease in the electron transport chain, and thus the consumption of O2 is drastically reduced.
Figure 4. Respiration parameters
Beyond the expected changes in respiration and ECAR, a number of respiratory parameters may be obtained from this data. This is summarized in the figure above:
Here we see that we may obtain information about the basal respiration of the cells, the percent of O2 consumption devoted to ATP production as well as the amount devoted to maintaining the proton gradient (due to H+ leak). Further, we may obtain the maximal respiratory rate under conditions of uncoupled respiration (sometimes referred to as spare respiratory capacity) and finally, we can determine the amount of O2 consumption not due to mitochondrial processes.
A rapidly growing number of studies are employing this mitochondrial profile to assess cellular bioenergetics, identify mitochondrial dysfunction and to predict the ability of cells to respond to stress and/or insults. For more information and details about this experimental method and the idea of spare respiratory capacity, please see refer to the following publications 1-8.
Min Wu, Per Bo Jensen, George W. Rogers, and David A. Ferrick are employees of Seahorse Bioscience, which produces reagents and instrumentation featured in this article.
|Oligomycin, FCCP, Rotenone and Antimycin A Solutions||Seahorse Bioscience||Seahorse Mito Stress Test Kit|
|DMEM Running Media||Seahorse Bioscience||100965-000|
|Distilled Water||GIBCO, by Life Technologies||15230-170|
|Calibration buffer||Seahorse Bioscience|