The authors would like to acknowledge the generous support of the John and Debbie Oxley Endowed Chair for Equine Sports Medicine and the Grayson Jockey Club Research Foundation.

This study was approved by the Oklahoma State University Institutional Animal Care and Use Committee. Four Thoroughbred geldings (17.5 &#177; 1.3 years, 593 &#177; 45 kg) were used in this study to generate the representative results.
1. Obtaining skeletal muscle biopsy specimen
<ol>
	<li>Obtain skeletal muscle biopsies (follow sterile technique) from the center of the semitendinosus muscle (or other muscle of interest), using a 12 G University College Hospital (UCH) biopsy ...

<ol>
	<li>Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+in+muscle+approaching+maximal+rates+of+oxygen+utilization.">Energy metabolism in muscle approaching maximal rates of oxygen utilization.</a> Medicine and Science in Sports and Exercise. 27 (1), 54-59 (1995).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mitochondrial Pathways and Respiratory Control. An Introduction to OXPHOS Analysis. 4th edn. , (2014).</li><li>Ehrenberg, B., Montana, V., Wei, M. D., Wuskell, J. P., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+potential+can+be+determined+in+individual+cells+from+the+nernstian+distribution+of+cationic+dyes.">Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.</a> Biophysical Journal. 53 (5), 785-794 (1988).</li><li>Chinopoulos, C., Kiss, G., Kawamata, H., Starkov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+ADP-ATP+exchange+in+relation+to+mitochondrial+transmembrane+potential+and+oxygen+consumption.">Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption.</a> Methods in Enzymology. 542, 333-348 (2014).</li><li>Krumschnabel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-resolution+measurement+of+mitochondrial+respiration+and+hydrogen+peroxide+production.">Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production.</a> Methods in Molecular Biology. 1264, 245-261 (2015).</li><li>Lemieux, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+function+is+altered+in+horse+atypical+myopathy.">Mitochondrial function is altered in horse atypical myopathy.</a> Mitochondrion. 30, 35-41 (2016).</li><li>Houben, R., Leleu, C., Fraipont, A., Serteyn, D., Votion, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+muscle+mitochondrial+respiratory+capacity+in+Standardbred+racehorses+as+an+aid+to+predicting+exertional+rhabdomyolysis.">Determination of muscle mitochondrial respiratory capacity in Standardbred racehorses as an aid to predicting exertional rhabdomyolysis.</a> Mitochondrion. 24, 99-104 (2015).</li><li>Votion, D. M., Gnaiger, E., Lemieux, H., Mouithys-Mickalad, A., Serteyn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+fitness+and+mitochondrial+respiratory+capacity+in+horse+skeletal+muscle.">Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle.</a> PLoS One. 7 (4), 34890 (2012).</li><li>Votion, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+mitochondrial+respiratory+function+in+response+to+endurance+training+and+endurance+racing.">Alterations in mitochondrial respiratory function in response to endurance training and endurance racing.</a> Equine Veterinary Journal Supplement. (38), 268-274 (2010).</li><li>Tosi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mitochondrial+oxidative+phosphorylation+capacity+in+horses+suffering+from+polysaccharide+storage+myopathy.">Altered mitochondrial oxidative phosphorylation capacity in horses suffering from polysaccharide storage myopathy.</a> Journal of Bioenergetics and Biomembranes. 50 (5), 379-390 (2018).</li><li>Davis, M. S., Fulton, M. R., Popken, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hyperthermia+and+acidosis+on+equine+skeletal+muscle+mitochondrial+oxygen+consumption.">Effect of hyperthermia and acidosis on equine skeletal muscle mitochondrial oxygen consumption.</a> Comparative Exercise Physiology. 17 (2), 171-179 (2021).</li><li>Latham, C. M., Fenger, C. K., White, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAPID+COMMUNICATION:+Differential+skeletal+muscle+mitochondrial+characteristics+of+weanling+racing-bred+horses1.">RAPID COMMUNICATION: Differential skeletal muscle mitochondrial characteristics of weanling racing-bred horses1.</a> Journal of Animal Science. , (2019).</li><li>White, S. H., Warren, L. K., Li, C., Wohlgemuth, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submaximal+exercise+training+improves+mitochondrial+efficiency+in+the+gluteus+medius+but+not+in+the+triceps+brachii+of+young+equine+athletes.">Submaximal exercise training improves mitochondrial efficiency in the gluteus medius but not in the triceps brachii of young equine athletes.</a> Scientific Reports. 7 (1), 14389 (2017).</li><li>White, S. H., Wohlgemuth, S., Li, C., Warren, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+communication:+Dietary+selenium+improves+skeletal+muscle+mitochondrial+biogenesis+in+young+equine+athletes.">Rapid communication: Dietary selenium improves skeletal muscle mitochondrial biogenesis in young equine athletes.</a> Journal of Animal Science. 95 (9), 4078-4084 (2017).</li><li>Doerrier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+FluoRespirometry+and+OXPHOS+protocols+for+human+cells,+permeabilized+fibers+from+small+biopsies+of+muscle,+and+isolated+mitochondria.">High-resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria.</a> Methods in Molecular Biology. 1782, 31-70 (2018).</li><li>Li, C., White, S. H., Warren, L. K., Wohlgemuth, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+aging+on+mitochondrial+function+in+skeletal+muscle+of+American+American+Quarter+Horses.">Effects of aging on mitochondrial function in skeletal muscle of American American Quarter Horses.</a> Journal of Applied Physiology. 121 (1), 299-311 (2016).</li><li>Komlodi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mitochondrial+incubation+media+for+measurement+of+respiration+and+hydrogen+peroxide+production.">Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production.</a> Methods in Molecular Biology. 1782, 137-155 (2018).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+physiology.">Mitochondrial physiology.</a> Bioenergetic Communications. , (2020).</li><li>Li Puma, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+oxygen+concentration+influences+rates+of+mitochondrial+hydrogen+peroxide+release+from+cardiac+and+skeletal+muscle+preparations.">Experimental oxygen concentration influences rates of mitochondrial hydrogen peroxide release from cardiac and skeletal muscle preparations.</a> American Journal of Physiology: Regulatory, Integrated, and Comparative Physiology. 318 (5), 972-980 (2020).</li></ol>

The authors have no conflicts of interest related to this manuscript.

The addition of fluorescent signals to the standard output of the high-resolution respirometer provides valuable information regarding mitochondrial physiology, but meticulous calibration of the fluorescent signal is critical for quality data. The original protocols for the use of MgG suggest that the calibration curves generated while calculating magnesium-adenylate dissociation constants could be applied to subsequent assays4; however, the fluorescent signal from the MgG may not be not sufficien...

The mitochondria of eukaryotic cells produce the majority of the ATP used by the cells for work and maintenance1. A key step in the mitochondrial production of ATP is the conversion of oxygen to water, and thus the metabolic capacity of mitochondria and the associated cells is frequently quantified through the measurement of oxygen consumption2. However, mitochondrial physiology is more complex than the simple process of oxygen consumption, and reliance on this endpoint exclusively provides an incomplete assessment of the impact of mitochondrial function and dysfunction on cellular health. Full characterization of mitochondrial function requires the assessment of not only oxygen consumption, but also the production of ATP as well as reactive oxygen species (ROS).
Additional measures of key mitochondrial functions can be accomplished concurrently with the measurement of respiration through the use of specific fluorophores. Tetramethylrhodamine methylester (TMRM) is a cationic fluorophore that accumulates in the mitochondrial matrix in proportion to the mitochondrial transmembrane voltage potential, resulting in a decrease in fluorescent intensity due to this accumulation3. TMRM can be used as an indicator of relative changes in mitochondrial membrane potential, or can be used to quantify precise changes in transmembrane voltage with additional experiments to determine constants that allow conversion of the fluorescent signal to mV. Magnesium green (MgG) is a fluorophore that fluoresces when bound with Mg2+, and is used for measurements of ATP synthesis based on the differential affinity of ADP and ATP for magnesium divalent cation4. Investigators must determine the specific affinity/dissociation constants (Kd) for both ADP and ATP under specific analytical conditions to convert the changes in MgG fluorescence to a change in ATP concentration. Amplex UltraRed (AmR) is the fluorophore used to measure the production of hydrogen peroxide and other ROS&#160;during mitochondrial respiration5. The reaction between H2O2 and AmR (which is catalyzed by horseradish peroxidase) produces resorufin, which is detectable through fluorescence at 530 nM. Each of these assays can be added individually to assays of real-time mitochondrial respiration, to provide concurrent measurements of the respective aspects of mitochondrial physiology, thus providing a direct link between respiration and mitochondrial output.
Horses are capable of very high rates of mass-specific oxygen consumption, due in part to the very high mitochondrial content of equine skeletal muscle, making this tissue highly relevant for studying mitochondrial physiology. With the development of high-resolution respirometry, studies using this novel technology have helped define the contributions of equine skeletal muscle mitochondria to both the remarkable exercise capacity of horses and the pathophysiology of skeletal muscle diseases6,7,8,9,10,11,12,13,14. Studies of equine skeletal muscle mitochondrial function are particularly advantageous, as obtaining large amounts of this tissue is non-terminal. Thus, equine subjects can not only provide sufficient tissue for the complete characterization of mitochondrial function, but also serve as longitudinal controls for high-quality, mechanistic studies into mitochondrial physiology. For this reason, additional assays to quantify mitochondrial membrane potential, ATP synthesis, and the production of ROS&#160;that complement the measurement of oxygen consumption in this tissue have been developed, in order to provide a more robust characterization of mitochondrial physiology in equine skeletal muscle.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ADP</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>A5285</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed</td>
			<td>Life Technologies</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>A6003</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium carbonate</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>C4830</td>
			<td></td>
		</tr>
		<tr>
			<td>CCCP</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>C2759</td>
			<td></td>
		</tr>
		<tr>
			<td>DatLab 7.0</td>
			<td>Oroboros Inc</td>
			<td></td>
			<td>Software to operate O2K fluororespirometer</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>DTPA</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>D1133</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>G1626</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>H7523</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>P8250</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>516813</td>
			<td>Must be made fresh daily prior to assay</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>K-MES</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>M8250</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Green</td>
			<td>Thermo Fisher Scientific</td>
			<td>M3733</td>
			<td></td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>M1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>M9647</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondrial isolation kit</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>MITOISO1</td>
			<td></td>
		</tr>
		<tr>
			<td>O2K fluororespirometer</td>
			<td>Oroboros Inc</td>
			<td></td>
			<td>Multiple units required to run full spectrum of assays concurrently.</td>
		</tr>
		<tr>
			<td>Phosphocreatine</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>P7936</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>P1767</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium lactobionate</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>L2398</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>P2256</td>
			<td>Must be made fresh daily prior to assay</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>S2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>84097</td>
			<td></td>
		</tr>
		<tr>
			<td>Superoxide dismutase</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>S8160</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Titration pump</td>
			<td>Oroboros Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titration syringes</td>
			<td>Oroboros Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TMRM</td>
			<td>Sigma-Aldrich (MilliporeSigma)</td>
			<td>T5428</td>
			<td></td>
		</tr>
		<tr>
			<td>UCH biopsy needle</td>
			<td>Millenium Surgical Corp</td>
			<td>72-238067</td>
			<td>Available in a range of sizes</td>
		</tr>
	</tbody>
</table>

high-resolution fluoro-respirometry of equine skeletal muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

The proposed reference state is that of a healthy sedentary Thoroughbred (no increased fitness due to compulsory exercise) and a fresh muscle sample collected from the center of a postural muscle, containing a high percentage of mitochondria-rich type I skeletal muscle fibers and incubated under conditions approximating resting metabolism (i.e., 38 &#176;C and pH 7.0). Under these conditions, the investigator can expect LN values of 2.71 &#177; 0.90, PN values of 62.40 &#177; 26.22, PN+S ...

Watch this Scientific Journal Video about High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle at JoVE.com

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...

These protocols allow the investigators to go beyond simply measuring mitochondrial oxygen consumption, and instead permit the measurement of key mitochondrial end products, ATP and reactive oxygen species. The main advantage of this technique is that the investigator can measure mitochondrial efficiency by directly comparing the rate of oxygen consumption to the rate of end product production. Whether the end product is ATP or reactive oxygen.The techniques require very careful attention to detail, particularly in avoiding bubbles. Whether they're in the respiration chamber or titration syringes, bubbles are your enemy because they make the measurements less precise. After obtaining the skeletal muscle biopsies.fill the respirometer chambers with magnesium-free media and seal the incubation chamber. Set the instrument incubation temperature to 38 degrees Celsius to represent the basal temperature of equine skeletal muscle. And set the mixing of the respiration media to 750 rpm using a magnetic stir turning in the bottom of the respirometer chamber.Next, turn off the chamber illumination to avoid interference with fluorescent sensors. Energize the oxygen electrode with an 800 millivolts polarization voltage and amplify the resulting signal with a gain setting of one. Record the oxygen concentration every two seconds and calculate the oxygen flux as the negative slope of the oxygen measurement over the proceeding 40 seconds.Then calibrate the oxygen sensor by allowing the media to equilibrate with room air. Calculate the reference oxygen partial pressure based on barometric pressure measured by the high resolution respirometer and standard atmospheric oxygen concentration. Use green fluorescent sensors to quantify the fluorescent signal from the respiration chamber.Energize the sensors at 400 to 500 millivolts and the resulting signal gets amplified with a gain of 1 to 1, 000. Next, add TMRM to the respiration chamber before adding mitochondria and calibrate the fluorescent signal using a simple two point calibration of the fluorescent signal versus the amount of added fluorophore before the addition of the mitochondria. Perform the final calibration of the TMRM signal after completion of the respirometry titration protocol by delivering several titrations of uncoupling agent until no further increases in TMRM fluorescent signal are observed indicating the complete collapse of mitochondrial membrane potential.Use blue fluorescent sensors to quantify the fluorescent signal from the respiration chamber and energize these sensors for individual instruments to capture the expected signal within the linear range of the sensor. Add eight microliters of two millimolar EDTA to the respiratory chamber to chelate cations that would compete with magnesium ions for binding to magnesium green. Then, add four microliters of one millimolar magnesium green to the respiration chamber.Calibrate the raw fluorescent signal with 10 by 2 microliters sequential titrations of 100 millimolar magnesium chloride, allowing one minute between titrations to stabilize the fluorescent signal. Determine the ATTP synthesis rate which is the slope of the concentration of ATP over time throughout the protocol. Use green fluorescent sensors to quantify the fluorescent signal from the respiration chamber.Energize the sensors at 300 to 400 millivolts. And the resulting signal gets amplified with a gain of 1 to 1, 000. Optimize specific settings for individual instruments to capture the expected signal within the linear range of the sensor.Before adding the mitochondria perform the chemical setup and initial calibration of the Amplex UltraRed Assay. Add 30 micromoles of DTPA to chelate cations that might interfere with the reaction. Then add superoxide dismutase or serratus peroxidase and Amplex UltraRed in the respirometry chamber.Allow the fluorescent signal to stabilize. Then add 0.2 micromoles of hydrogen peroxide twice with a gap of five minutes. Perform additional two point calibrations throughout the assay to allow adjustment of the responsiveness of the assay as the chemistry of the respirometry changes throughout the assay with the specific timing of these calibration points at the investigator's discretion.Vortex the sample to maintain a uniform sample suspension and add 15 microliters of isolated mitochondria suspension to each two milliliter incubation chamber so that the results represent the mitochondrial yield of 18.75 milligrams of muscle. Before adding any substrates, measure the residual oxygen consumption and subtract this value from the oxygen consumption values of each step in the substrate uncoupled inhibitor titration or SUIT protocol after completion. Use a general purpose SUIT that allows for the initial characterization of equine skeletal muscle mitochondrial function.Start with sequential titrations of pyruvate, glutamate, and malate into each other into each chamber to produce nicotinamide adenine dinucleotide and stimulate nonphosphorylating respiration supported by NADH oxidized through complex one-based leak. Then add ADP to stimulate phosphorylating respiration through complex one phosphorylating respiration. Add succinate to produce phosphorylating respiration by combining complex one and complex two phosphorylating respiration Add rotenone to block complex one.The resulting oxygen flux represents the capacity of complex two to support mitochondrial oxygen consumption by the oxidation of succinate alone. A high resolution respirometry trace of equine skeletal muscle mitochondrial respiration and relative membrane potential is shown. Respiration values of mitochondria incubated with TMRM are lower due to an inhibitory effect of that fluorophore.Mitochondrial respiration and ATTP synthesis of equine skeletal muscle containing a high percentage of mitochondria rich type one skeletal muscle fibers and incubated under conditions approximating resting metabolism are shown. Respirometry trace of equine skeletal muscle mitochondrial respiration and production of hydrogen peroxide is shown. High resolution respirometry requires a lot of patience.The person running the assay will have numerous time points at which they need to make a judgment call regarding whether a steady state has been reached and the next step can occur. We've only demonstrated a single titration protocol. There are dozens more protocols that can be applied in the same way to address specific questions regarding mitochondrial metabolism.

high-resolution-fluoro-respirometry-of-equine-skeletal-muscle

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Biology

1.1K visualizações.  Oklahoma State University. Esses protocolos permitem que os pesquisadores vão além de simplesmente medir o consumo de oxigênio mitocondrial e, em vez disso, permitem a medição dos principais produtos finais mitocondriais, ATP e espécies reativas de oxigênio. A principal vantagem dessa técnica é que o investigador pode medir a eficiência mitocondrial comparando diretamente a taxa de consumo de oxigênio com a taxa de produção do produto final. Se o produto final é ATP ou oxigênio reativo.As técnica...