Name: measurement of mitochondrial oxygen consumption in permeabilized fibers of drosophila using minimal amounts of tissue
Uploaded: 2018-04-07T14:00:00
Description: Watch this Scientific Journal Video about Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue at JoVE.com

This study was funded by grants from the National Sciences and Engineering Research Council (NSERC, discovery grant) and Universit&#233; de Moncton to NP. LHB would like to acknowledge the funding support from the Canadian Institute of Health Research (CIHR), the New Brunswick Innovation Foundation (NBIF) and Universit&#233; de Moncton. The work of EHC is supported by the Alzheimer Society of Canada, Brain Canada, NSERC, Canadian Breast Cancer Foundation, New Brunswick Innovation Foundation, New Brunswick Health Research Foundation and Universit&#233; de Moncton.

1. Reagents Preparation
<ol>
	<li>Prepare the following solutions for dissection and permeabilization of the tissue.
	<ol>
		<li>Prepare preservation solution: 2.77 mM CaK2EGTA, 7.23 mM K2EGTA, 5.77 mM Na2ATP, 6.56 mM MgCl2, 20 mM taurine, 15 mM Na2phosphocreatine, 20 mM imidazole, 0.5 mM dithiothreitol, and 50 mM K-MES, pH 7.1 (can be stored at -20 &#176;C).</li>
		<li>Prepare saponin solution: 5 mg of saponin in 1 mL of preservation solution (prepare fresh daily)...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+substrate+specificity+in+beetle+flight+muscle:+assessing+respiratory+oxygen+flux+in+small+samples+from+Dermestes+maculatus+and+Tenebrio+molitor.">Mitochondrial substrate specificity in beetle flight muscle: assessing respiratory oxygen flux in small samples from Dermestes maculatus and Tenebrio molitor.</a> Physiological Entomology. 41 (2), 96-102 (2016).</li><li>Teulier, L., Weber, J. M., Crevier, J., Darveau, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proline+as+a+fuel+for+insect+flight:+enhancing+carbohydrate+oxidation+in+hymenopterans.">Proline as a fuel for insect flight: enhancing carbohydrate oxidation in hymenopterans.</a> Proceedings of the Royal Society of London B: Biological Sciences. 283 (1834), 20160333 (2016).</li><li>Ferguson, M., Mockett, R. J., Shen, Y., Orr, W. C., Sohal, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+decline+in+mitochondrial+respiration+and+electron+transport+in+Drosophila+melanogaster.">Age-associated decline in mitochondrial respiration and electron transport in Drosophila melanogaster.</a> The Biochemical journal. 390 (2), 501-511 (2005).</li><li>Miwa, S., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+topology+of+superoxide+production+by+complex+III+and+glycerol+3-phosphate+dehydrogenase+in+Drosophila+mitochondria.">The topology of superoxide production by complex III and glycerol 3-phosphate dehydrogenase in Drosophila mitochondria.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1709 (3), 214-219 (2005).</li><li>Katewa, S. D., Ballard, J. W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympatric+Drosophila+simulans+flies+with+distinct+mtDNA+show+difference+in+mitochondrial+respiration+and+electron+transport.">Sympatric Drosophila simulans flies with distinct mtDNA show difference in mitochondrial respiration and electron transport.</a> Insect Biochemistry and Molecular Biology. 37 (3), 213-222 (2007).</li><li>Brand, M. D., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochemical Journal. 435 (2), 297-312 (2011).</li><li>St-Pierre, J., Buckingham, J. A., Roebuck, S. J., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology+of+superoxide+production+from+different+sites+in+the+mitochondrial+electron+transport+chain.">Topology of superoxide production from different sites in the mitochondrial electron transport chain.</a> Journal of Biological Chemistry. 277 (47), 44784-44790 (2002).</li><li>Kuznetsov, A. V., Veksler, V., Gellerich, F. N., Saks, V., Margreiter, R., Kunz, W. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+isolation,+structure+and+function.">Mitochondria: isolation, structure and function.</a> The Journal of Physiology. 589 (18), 4413-4421 (2011).</li><li>Pichaud, N., Ballard, J. W. O., Tanguay, R. M., Blier, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+sensitivity+of+mitochondrial+functions+in+permeabilized+muscle+fibers+from+two+populations+of+Drosophila+simulans+with+divergent+mitotypes.">Thermal sensitivity of mitochondrial functions in permeabilized muscle fibers from two populations of Drosophila simulans with divergent mitotypes.</a> American Journal of Physiology - Regulatory, Integrative and Comparative Physiology. 301 (1), R48-R59 (2011).</li><li>Pichaud, N., Ballard, J. W. O., Tanguay, R. M., Blier, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+mitochondrial+dna+haplotypes+exhibit+metabolic+differences:+insight+into+functional+properties+of+mitochondria.">Naturally occurring mitochondrial dna haplotypes exhibit metabolic differences: insight into functional properties of mitochondria.</a> Evolution. 66 (10), 3189-3197 (2012).</li><li>Pichaud, N., Messmer, M., Correa, C. C., Ballard, J. W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+influences+the+intake+target+and+mitochondrial+functions+of+Drosophila+melanogaster+males.">Diet influences the intake target and mitochondrial functions of Drosophila melanogaster males.</a> Mitochondrion. 13 (6), 817-822 (2013).</li><li>Wolff, J. N., Pichaud, N., Camus, M. F., C&#244;t&#233;, G., Blier, P. U., Dowling, D. 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P., Pandhare, J., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolism+of+proline,+a+stress+substrate,+modulates+carcinogenic+pathways.">The metabolism of proline, a stress substrate, modulates carcinogenic pathways.</a> Amino Acids. 35 (4), 681-690 (2008).</li><li>Bender, T., Martinou, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+pyruvate+carrier+in+health+and+disease:+To+carry+or+not+to+carry?.">The mitochondrial pyruvate carrier in health and disease: To carry or not to carry?.</a> Biochimica et Biophysica Acta - Molecular Cell Research. 1863 (10), 2436-2442 (2016).</li><li>Divakaruni, A. 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In this study, a method for sample preparation prior to the measurements of mitochondrial oxygen consumption in Drosophila is described. This method was developed to overcome different problems related to the protocols using mitochondrial isolations, notably in terms of duration and number of individuals required. Instead of working with mitochondrial isolations usually requiring large amount of tissues obtained from several individuals, this experiment is performed on permeabilized muscle fibers from thoraxes of few Dro...

The fruit fly, Drosophila melanogaster, has been used as a model organism for genetic research for over a century1. The study of this organism has not only led to significant fundamental knowledge about sex-linked inheritance2, mutation rate3, the development of neural system and the cell fate determination4, but has also recently emerged as a valuable tool to study the mechanisms inherent to several diseases such as Alzheimer&#39;s and Parkinson&#39;s5,6. Moreover, it is a popular model to study the aging process, as they can be raised in large number over a short period of time and have a short lifespan. They also possess homologous structures to human organs, such as a heart, oenocytes (hepatocyte-like cells), fat bodies (functioning similarly as the liver and white adipose tissue), insulin producing cells (equivalent to &#946;-pancreatic cells), as well as the hemolymph transporting metabolites (analogous to the blood of vertebrates)7. Moreover, the central pathways of intermediary metabolism (including the insulin/insulin-like growth factor-like signaling pathway and Target of Rapamycin-TOR pathways) are also highly conserved7. For these reasons, Drosophila have recently been exploited to describe the fundamental mechanisms that control metabolism, especially in pathological conditions inherent to human metabolic diseases such as diabetes8. A major component of the metabolism is the mitochondrion that integrates multiple pathways and performs one of life&#39;s most important biological functions, ATP production, via the oxidative phosphorylation process (OXPHOS). Considering their central role in organism&#39;s metabolism, it is not surprising that mitochondrial dysfunctions are involved in many diseases like Parkinson&#39;s9 and Alzheimer&#39;s diseases10, as well as in amyotrophic lateral sclerosis11,12. They are also fundamental determinants of the aging process. Indeed, they are the main producers of reactive oxygen species (ROS) in the cell, which can be detrimental to the cell at high concentration through oxidative damages11. Aging has also been associated to the accumulation of damaged or mutated mitochondrial DNA13, mitophagy dysfunctions14,15 as well as impairment of mitochondrial biogenesis16. Mitochondria are also key determinants of the cell&#39;s homeostasis as they can utilize different substrates to adjust several cellular functions according to the abundance or scarcity of macronutrients17,18.
Indeed, the different nutrients in the diet (carbohydrates, lipids and proteins) are digested, absorbed and transported in the cells. They are then transformed in the cytosol, and the derived substrates are transported into the mitochondrial matrix where they produce reducing equivalents, such as NADH and FADH219. These reducing equivalents are then oxidized by different enzymatic complexes of the electron transport system (ETS). These complexes are embedded in the mitochondrial inner membrane, such as Complex I and Complex II. Additionally, other enzymatic complexes such as the mitochondrial glycerol-3-phosphate dehydrogenase and the proline dehydrogenase represent alternate routes for the entry of electrons into the ETS20,21. These &#39;alternative&#39; complexes are particularly important in insects, as according to the species, they can actively participate to increase the respiration20,22,23,21. Electrons from these ETS feeding systems are transferred to the ubiquinone and subsequently to Complex III, and then to Complex IV, until the final acceptor, molecular oxygen. This electron transfer generates a proton-motive force across the inner mitochondrial membrane driving the phosphorylation of ADP to ATP at Complex V (Figure 1). Considering the central role of mitochondria in cell&#39;s homeostasis, studying mitochondrial metabolism using the relevant model D. melanogaster represents a powerful tool to delineate the underlying mechanisms of various pathophysiological conditions or under cellular and environmental stresses. Surprisingly however, only a handful of studies actually measured mitochondrial respiration in Drosophila24,25,26. Indeed, experiments aiming to evaluate mitochondrial oxygen consumption require the isolation of mitochondria. Although advantageous for the measurement of different mitochondrial functions (such as ROS production or P/O ratio as marker of mitochondrial efficiency27,28), these isolations generally require rather large quantities of tissue from several individuals24,29. This requirement for high amounts of tissue and individuals is an important limiting factor, especially considering that all individuals should be the same age and preferably of the same sex for the experiments, making the measure of respiration at different time points laborious at best. Moreover, while mitochondrial isolations can provide significant insight into the fundamental mechanisms governing mitochondrial metabolism, the methods used to isolate mitochondria have several drawbacks such as the difficulty to obtain replicable results, disruption of the mitochondrial network, and alteration of mitochondrial structure and function29,30,31.
The aim of this study is to present a robust protocol to measure mitochondrial oxygen consumption in Drosophila using only a minimal amount of tissue from very few individuals. This protocol consists of measuring mitochondrial oxygen consumption in situ using permeabilized muscle fibers29 from Drosophila thoraxes in combination with high-resolution respirometry32,33,34,35. This method has also additional advantages compared to the classic mitochondrial isolation method since the interactions with the other components of the cell as well as mitochondrial structure and function are more preserved in permeabilized fibers29,31,36, which makes this approach more physiologically relevant. With this protocol, mitochondrial functions can be accurately evaluated using high-resolution respirometry in only three thoraxes of Drosophila, with substrates allowing the determination of oxygen consumption at several different steps of the ETS. Therefore, this protocol could help answer key questions about the fundamental mechanisms that control metabolism in the context of many environmental or pathophysiological conditions by taking advantage of the Drosophila model.
To measure the oxygen consumption at several different steps of the ETS and evaluate how different substrates contribute to the respiration, different substrates (Figure 1), uncoupler, and inhibitors are used30 after permeabilization of the tissue. Specifically, sequential additions of different substrates are performed to stimulate the entry of electrons through different complexes of the ETS. An uncoupler, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), is then added at optimal concentration to measure the non-coupled respiration, i.e., the non-phosphorylating respiration stimulated to maximal oxygen consumption. Sequential inhibitions of complexes I, II, and III are then performed to monitor the residual oxygen consumption that is due to non-ETS oxidation reactions. Finally, complex IV maximal respiration capacity can be evaluated by the injection of N,N,N&#39;,N,-Tetramethyl-p-phenylenediamine (TMPD), an artificial electron provider, and ascorbate. It is important to note that the experiments are conducted at 24 &#176;C since it is the temperature at which the flies are raised.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>High-resolution respirometer Oxygraph O2K</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>10022-02</td>
			<td>Startup O2K respirometer kit 
			&#160;</td>
		</tr>
		<tr>
			<td>O2K-Titration Set&#160;</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>20820-03</td>
			<td>Hamilton syringes with different volumes 
			&#160;</td>
		</tr>
		<tr>
			<td>Datlab software</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>20700</td>
			<td>Software for data acquisition and analysis 
			&#160;</td>
		</tr>
		<tr>
			<td>Fine-tipped antimagnetic forceps</td>
			<td>VWR</td>
			<td>82027-400</td>
			<td> 
			&#160;</td>
		</tr>
		<tr>
			<td>Secura225D-1S-DQE</td>
			<td>Sartorius AG, Goettingen, Germany</td>
			<td></td>
			<td>Semi-micro balance (distributed by several companies)&#160; 
			&#160;</td>
		</tr>
		<tr>
			<td>Drosophila melanogaster wild-type w1118</td>
			<td>Bloomington Drosophila stock Center, IN, USA</td>
			<td></td>
			<td> 
			Storage Condition: 24 &#176;C</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>E4378</td>
			<td>EGTA 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma-Aldrich</td>
			<td>P1767</td>
			<td>CAUTION: corrosive to metals, acute toxicity, skin corrosion, serious eye damage, acute aquatic toxicity. 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>CaCO3</td>
			<td>Sigma-Aldrich</td>
			<td>C4830</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td> 
			Storage Condition: -20 &#176;C</td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Na2Phosphocreatine</td>
			<td>Sigma-Aldrich</td>
			<td>P7936</td>
			<td> 
			Storage Condition: -20 &#176;C</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td> 
			Storage Condition: 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M8250</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Saponin from quillaja bark</td>
			<td>Sigma-Aldrich</td>
			<td>S7900</td>
			<td>Saponin 
			Storage Condition: RT 
			Solution Preparation: 5 mg in 1 mL of preservation solution. Prepare fresh daily.</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>05470</td>
			<td> 
			Storage Condition: 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>Na2S2O4</td>
			<td>Sigma-Aldrich</td>
			<td>157953</td>
			<td>Sodium dithionite. CAUTION: self-heating substances and mixtures, acute toxicity, acute aquatic toxi chronic aquatic toxicity. 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256</td>
			<td>Pyruvate 
			Storage Condition: 2-8 &#176;C 
			Solution Preparation: In MilliQ water. Prepare fresh daily.</td>
		</tr>
		<tr>
			<td>L-(-)-Malic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M1000</td>
			<td>Malate 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Neutralize with KOH and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-diphosphate monopotassium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A5285</td>
			<td>ADP 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In MilliQ water. Neutralize with KOH and store at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>Cytochrome c from equine heart</td>
			<td>Sigma-Aldrich</td>
			<td>C7752</td>
			<td>Cytochrome c 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In MilliQ water. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma-Aldrich</td>
			<td>P0380</td>
			<td>Proline 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Sodium succinate dibasic hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S2378</td>
			<td>Succinate 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Neutralize with HCl and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>sn-Glycerol 3-phosphate bis(cyclohexylammonium) salt</td>
			<td>Sigma-Aldrich</td>
			<td>G7886</td>
			<td>Glycerol-3-phosphate 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In MilliQ water. Neutralize with HCl and store at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td>FCCP. CAUTION: acute toxicity, skin sensitisation, chronic aquatic toxicity. 
			Storage Condition: RT 
			Solution Preparation: In absolute ethanol. Store in glass vials at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td>CAUTION: acute toxicity, skin irritation, eye irritation, specific target organ toxicity (respir sytem), acute aquatic toxicity, chronic aquatic toxicity. 
			Solution Preparation: In absolute ethanol. Store in dark vials at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Malonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M1296</td>
			<td>Malonate. CAUTION: acute toxicity, serious eye damage. 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Neutralize with KOH. Prepare fresh daily.</td>
		</tr>
		<tr>
			<td>Antimycin A from Streptomyces sp.</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td>Antimycin A. CAUTION: acute toxicity, acute aquatic toxicity, chronic aquatic toxicity. 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In absolute ethanol. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethyl-p-phenylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>T7394</td>
			<td>TMPD 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Store in dark vials at&#160; -20 &#176;C.</td>
		</tr>
		<tr>
			<td>(+)-Sodium L-ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td>Ascorbate 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Store in dark vials at&#160; -20 &#176;C.</td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>Sodium azide. CAUTION: acute toxicity (oral and dermal), specific target organ toxicity (brain), aquatic toxicity, chronic aquatic toxicity.&#160; 
			Solution Preparation: In MilliQ water. Store at -20 &#176;C.</td>
		</tr>
	</tbody>
</table>

measurement of mitochondrial oxygen consumption in permeabilized fibers of drosophila using minimal amounts of tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

A representative trace of mitochondrial oxygen consumption using the protocol described above is provided in Figure 2. The pyruvate and malate injected in the chambers along with the permeabilized muscle fibers are referred to the CI-LEAK respiration, i.e., when the complex I of the ETS is stimulated by the NADH produced through oxidation of pyruvate and malate via the tricarboxylic acid cycle (CI). During this respiration rate, the mitochondrial oxy...

Watch this Scientific Journal Video about Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue at JoVE.com

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

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