The authors thank the staff members of the Department of Physiology in the Faculty of Medicine in Hradec Kr&#225;lov&#233; and the Department of Pathophysiology in the Third Faculty of Medicine for the help with chemicals and samples preparation. This work was supported by Charles University grant programs PROGRES Q40/02, Czech Ministry of Health grant NU21-01-00259, Czech science foundation grant 18-10144 and INOMED project CZ.02.1.01/0.0/0.0/18_069/0010046 funded by the Ministry of Education, Youth and Sports of the Czech Republic and by the European Union.

1. One day before the assay
<ol>
	<li>Preparation of reagents and substrates.
	<ol>
		<li>Mitochondrial assay solution (MAS): Prepare stock solutions of all reagents as described in Table 1. Warm the stocks of mannitol and sucrose to 37 &#176;C to dissolve completely. Mix the reagents to prepare 2x MAS, then warm the mixture to 37 &#176;C. Adjust the pH with 5N KOH to 7.4 (~7 mL), then add water to bring the volume up to 1 L. Filter-sterilize&#160;and store the aliquots at -20 &#176;C until the measurement day.</li>
		<li>Bovine serum albumin (5% BSA): Dissolve 5 g of BSA in 90 mL of prewarmed sterile water on a magnetic stirrer and avoid shaking. Adjust the pH to 7.4 with 5 N KOH, and then add water to bring the volume up to 100 mL. Filter-sterilize and store the aliquots at -20 &#176;C until the measurement day.</li>
		<li>Mitochondrial substrates: Prepare 1 M stock solutions of sodium succinate, sodium pyruvate, and sodium glutamate in sterile water. Prepare 100 mM stock solution of sodium malate in sterile water and use prewarmed sterile water to prepare a 10 mM stock solution of&#160;palmitoyl carnitine. Adjust pH of each solution to 7.4 by 5 N KOH and filter-sterilize. Store the substrates at 2-8 &#176;C. At the time of use, warm palmitoyl carnitine to 37 &#176;C to dissolve any precipitates. For later use, store aliquots of all the substrates except pyruvate at -20 &#176;C.</li>
		<li>Mitochondrial inhibitors: Use dimethyl sulfoxide (DMSO) to prepare stock solutions of 25 mM oligomycin, 50 mM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), 20 mM rotenone, and 20 mM antimycin A. Store the aliquots at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Seeding and treating the cells: As shown in Figure 2, seed the cells in columns 2-11. Columns 1 and 12 must be left empty to serve as background wells. In this work, HepG2 and A549 cells were used.&#160;
	<ol>
		<li>Seed the cells at a density of 20,000 cells per well. Use 50-80 &#181;L of cell culture medium for seeding.</li>
		<li>Fill the blank wells with an equal volume of cell culture medium and incubate the cells at 37 &#176;C in a humidified incubator with 5% CO2. Allow the cells to attach for 3-4 hours, and then add 100 &#181;L of cell culture medium to all wells.</li>
		<li>With that final medium addition, apply the treatment in columns 7&#8211;11. In this work, the experimental group was treated with 1 mM metformin hydrochloride for 16 hours, and the control group was treated with an equal volume of sterile distilled water as a vehicle control.</li>
	</ol>
	</li>
	<li>Hydrating the sensors: Pipette 200 &#181;L of sterile water per well into the utility plate, then carefully return the sensor cartridge while immersing the sensors in water. Incubate the cartridge in a CO2-free incubator at 37 &#176;C till the next day.</li>
	<li>The assay protocol template: Switch on the analyzer (see Table of Materials) and the controller unit. Start the instrument control and data acquisition software and design the assay protocol as described in Table 2. Under Group Definitions, create four injection strategies where Port A differs according to the injected substrate (Figure 3). Name the strategies after the substrates or their abbreviations.</li>
	<li>To ports B, C, and D, assign the compounds&#160;oligomycin, FCCP, and rotenone/antimycin A, respectively. Create eight groups and name them as shown in Figure 4. Under Plate Map assign the groups to the corresponding wells, then save the protocol as a ready-to-use template. Leave the analyzer switched on to allow the temperature to stabilize overnight. Keep the analyzer in a place with a stable temperature to avoid sudden temperature changes.</li>
</ol>
2. The day of the assay
<ol>
	<li>Replacing water with the calibrant: Discard the water from the utility plate and pipette 200 &#181;L of prewarmed calibrant per well into the utility plate. Return the cartridge to the CO2-free incubator until the time of the assay. To avoid rapid evaporation of the calibrant, maintain a source of humidity inside the incubator and turn off or reduce the fan speed to a minimum.</li>
	<li>Preparing the working solutions: Start by warming 2x MAS, 5% BSA, and sterile water to 37 &#176;C. Meanwhile, allow the inhibitor stocks to reach room temperature. Use warm 2x MAS and sterile distilled water to prepare 5 mL of the working concentration of the substrates and inhibitors as described in Table 3.</li>
	<li>Loading the injection ports: As shown in Figure 3, load 20 &#181;L of the substrates into port A. Load succinate/rotenone mixture into port A of rows A and B. Load pyruvate/malate mixture into port A of rows C and D. Load glutamate/malate mixture into port A of rows E and F. Load palmitoyl carnitine/malate mixture into port A of rows G and H. For the whole plate, load port B with 22 &#181;L of oligomycin preparation, port C with 25 &#181;L of FCCP preparation, and port D with 27 &#181;L of rotenone/antimycin A mixture.</li>
	<li>Starting the calibration step: Under Run Assay tab, click on Start Run to start the assay. Insert the loaded sensor cartridge and start the calibration step. Wait for the calibration to complete before proceeding to the next step.</li>
	<li>Preparation of the assay medium (MAS-BSA-rPFO): To prepare 20 mL of the assay medium, mix 10 mL of 2x MAS, 9.2 mL of sterile water, and 0.8 mL of 5% BSA in a 50 mL tube. Add 2 &#181;L of 10&#160;&#181;M&#160;rPFO to attain a concentration of 1 nM and resuspend the mixture with gentle pipetting. Avoid shaking and do not use a vortex mixer for mixing. Incubate the tube at 37 &#176;C until the time of use.</li>
	<li>Washing the cells: Wash the cells and the empty blank wells two times with prewarmed calcium- and magnesium-free phosphate buffered saline (PBS) using a multichannel pipette. Avoid reusing the same tips to discard the cell culture medium and to add PBS. Perform this step outside the laminar flow to protect the cells from drying out by the airflow.</li>
	<li>Cell permeabilization in assay medium: Using a multichannel pipette, discard the PBS and replace it with 180 &#181;L of the prewarmed assay medium (MAS-BSA-rPFO).</li>
	<li>Starting the measurement: Immediately after permeabilization, replace the utility plate of the calibrated sensor cartridge with the cell plate&#160;and start the measurement.</li>
</ol>

<ol>
	<li>Gerencser, A. A., 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=Quantitative+microplate-based+respirometry+with+correction+for+oxygen+diffusion.">Quantitative microplate-based respirometry with correction for oxygen diffusion.</a> Analytical Chemistry. 81 (16), 6868-6878 (2009).</li><li>Murphy, E., 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,+biology,+and+role+in+disease:+A+scientific+statement+from+the+American+Heart+Association.">Mitochondrial function, biology, and role in disease: A scientific statement from the American Heart Association.</a> Circulation Research. 118 (12), 1960-1991 (2016).</li><li>Owen, O. E., Kalhan, S. C., Hanson, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+key+role+of+anaplerosis+and+cataplerosis+for+citric+acid+cycle+function.">The key role of anaplerosis and cataplerosis for citric acid cycle function.</a> Journal of Biological Chemistry. 277 (34), 30409-30412 (2002).</li><li>Nicholls, D. G., Ferguson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioenergetics 3. , (2002).</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>Sta&#328;kov&#225;, P., 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=Adaptation+of+mitochondrial+substrate+flux+in+a+mouse+model+of+nonalcoholic+fatty+liver+disease.">Adaptation of mitochondrial substrate flux in a mouse model of nonalcoholic fatty liver disease.</a> International Journal of Molecular Sciences. 21 (3), 1101 (2020).</li><li>Salabei, J. K., Gibb, A. A., Hill, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+measurement+of+respiratory+activity+in+permeabilized+cells+using+extracellular+flux+analysis.">Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis.</a> Nature Protocols. 9 (2), 421-438 (2014).</li><li>Divakaruni, A. S., 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=Thiazolidinediones+are+acute,+specific+inhibitors+of+the+mitochondrial+pyruvate+carrier.">Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (14), 5422-5427 (2011).</li><li>Divakaruni, A. S., Rogers, G. W., Murphy, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+function+in+permeabilized+cells+using+the+seahorse+XF+analyzer+or+a+Clark-type+oxygen+electrode.">Measuring mitochondrial function in permeabilized cells using the seahorse XF analyzer or a Clark-type oxygen electrode.</a> Current Protocols in Toxicology. 60, 1-16 (2014).</li><li>Elkalaf, M., T&#367;ma, P., Weiszenstein, M., Pol&#225;k, J., Trnka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+probe+Methyltriphenylphosphonium+(TPMP)+inhibits+the+Krebs+cycle+enzyme+2-Oxoglutarate+dehydrogenase.">Mitochondrial probe Methyltriphenylphosphonium (TPMP) inhibits the Krebs cycle enzyme 2-Oxoglutarate dehydrogenase.</a> PLoS One. 11 (8), 0161413 (2016).</li><li>Rogers, G. W., 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+throughput+microplate+respiratory+measurements+using+minimal+quantities+of+isolated+mitochondria.">High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.</a> PLoS One. 6 (7), 21746 (2011).</li></ol>

The authors have no conflict of interest to declare.

This protocol is a modification of previously published studies7,8,9,10 and the product user guide. In contrast to the manufacturer&#39;s protocol, 2x MAS is used instead of 3x MAS, since 2&#215; MAS is easier to dissolve and does not form precipitations after freezing. Frozen 2x MAS aliquots can be stored up to six months and show consistent results. Another difference is including ADP in the ...

Microplate-based respirometry has revolutionized mitochondrial research by enabling the study of cellular respiration of a small sample size1. Cellular respiration is generally considered as an indicator of mitochondrial function or &#39;dysfunction&#39;, despite the fact that the mitochondrial range of functions extends beyond energy production2. In aerobic conditions, mitochondria extract the energy stored in different substrates by breaking down and converting these substrates into metabolic intermediates that can fuel the citric acid cycle3 (Figure 1). The continuous flux of substrates is essential for the flow of the citric acid cycle to generate high energy &#39;electron donors&#39;, which deliver electrons to the electron transport chain that generates a proton gradient across the inner mitochondrial membrane, enabling ATP-synthase to phosphorylate ADP to ATP4. Therefore, an experimental design to assay mitochondrial respiration must include the sample nature (intact cells, permeabilized cells, or isolated mitochondria) and mitochondrial substrates.
Cells keep a store of indigenous substrates5, and mitochondria oxidize several types of substrates simultaneously6, which complicates the interpretation of results obtained from experiments performed on intact cells. A common approach to investigate mitochondrial ability to oxidize a selected substrate is to isolate mitochondria or permeabilize the investigated cells5. Although isolated mitochondria are ideal for quantitative studies, the isolation process is laborious. It faces technical difficulties such as the need for large sample size, purity of the yield, and reproducibility of the technique5. Permeabilized cells offer a solution for the disadvantages of mitochondrial isolation; however, routine permeabilizing agents of detergent nature are not specific and may damage mitochondrial membranes5.
Recombinant perfringolysin O (rPFO) was offered as a selective plasma membrane permeabilizing agent7, and it was used successfully in combination with an extracellular flux analyzer in several studies7,8,9,10. We have modified a protocol using rPFO to screen mitochondrial substrate flux using XFe96 extracellular flux analyzer. In this protocol, four different substrate oxidizing pathways in two cellular phenotypes are compared while having sufficient replicates and the proper control for each tested material.

<table><tbody><tr><td>Adenosine 5&amp;prime; -diphosphate monopotassium salt dihydrate</td><td>Merck</td><td>A5285</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Antimycin A</td><td>Merck</td><td>A8674</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Bovine serum albumin</td><td>Merck</td><td>A3803</td><td>store at 2 - 8 &amp;deg;C</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td><td>Merck</td><td>C2920</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Dimethyl sulfoxide</td><td>Merck</td><td>D8418</td><td>store at RT</td></tr><tr><td>D-Mannitol</td><td>Merck</td><td>63559</td><td>store at RT</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline</td><td>Gibco</td><td>14190-144</td><td>store at RT</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid</td><td>Merck</td><td>03777</td><td>store at RT</td></tr><tr><td>HEPES</td><td>Merck</td><td>H7523</td><td>store at RT</td></tr><tr><td>L(-)Malic acid disodium salt</td><td>Merck</td><td>M9138</td><td>store at RT</td></tr><tr><td>L-Glutamic acid sodium salt hydrate</td><td>Merck</td><td>G5889</td><td>store at RT</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Merck</td><td>M2670</td><td>store at RT</td></tr><tr><td>Oligomycin</td><td>Merck</td><td>O4876</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Palmitoyl-DL-carnitine chloride</td><td>Merck</td><td>P4509</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Potassium hydroxide</td><td>Merck</td><td>484016</td><td>store at RT</td></tr><tr><td>Potassium phosphate monobasic</td><td>Merck</td><td>P5655</td><td>store at RT</td></tr><tr><td>Rotenone</td><td>Merck</td><td>R8875</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Seahorse Wave Desktop Software</td><td>Agilent technologies</td><td></td><td>Download from www.agilent.com</td></tr><tr><td>Seahorse XFe96 Analyzer</td><td>Agilent technologies</td><td></td><td></td></tr><tr><td>Seahorse XFe96 FluxPak</td><td>Agilent technologies</td><td>102416-100</td><td>XFe96 sensor cartridges and XF96 cell culture microplates</td></tr><tr><td>Sodium pyruvate</td><td>Merck</td><td>P2256</td><td>store at 2 - 8 &amp;deg;C</td></tr><tr><td>Sodium succinate dibasic hexahydrate</td><td>Merck</td><td>S2378</td><td>store at RT</td></tr><tr><td>Sucrose</td><td>Merck</td><td>S7903</td><td>store at RT</td></tr><tr><td>Water</td><td>Merck</td><td>W3500</td><td>store at RT</td></tr><tr><td>XF calibrant</td><td>Agilent technologies</td><td>100840-000</td><td>store at RT</td></tr><tr><td>XF Plasma membrane permeabilizer</td><td>Agilent technologies</td><td>102504-100</td><td>Recombinant perfringolysin O (rPFO) - Aliquot and store at -20 &amp;deg;C</td></tr></tbody></table>

measuring mitochondrial substrate flux in recombinant perfringolysin o-permeabilized cells

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or enzymes are involved in the pathogenesis of several diseases. Mitochondrial substrate flux can be studied using intact cells, permeabilized cells, or isolated mitochondria. Investigating intact cells encounters several problems due to simultaneous oxidation of different substrates. Besides, several cell types contain internal stores of different substrates that complicate results interpretation. Methods such as mitochondrial isolation or using permeabilizing agents are not easily reproducible. Isolating pure mitochondria with intact membranes in sufficient amounts from small samples is problematic. Using non-selective permeabilizers causes various degrees of unavoidable mitochondrial membrane damage. Recombinant perfringolysin O (rPFO) was offered as a more appropriate permeabilizer, thanks to its ability to selectively permeabilize plasma membrane without affecting mitochondrial integrity. When used in combination with microplate respirometry, it allows testing the flux of several mitochondrial substrates with enough replicates within one experiment while using a minimal number of cells. In this work, the protocol describes a method to compare mitochondrial substrate flux of two different cellular phenotypes or genotypes and can be customized to test various mitochondrial substrates or inhibitors.

Start by normalizing the results to the second measurement of baseline respiration to show values as oxygen consumption rate percentage (OCR%). The results of the assay are shown in Figures 5, Figure 6, Figure 7, and Figure 8. It is important to assign the proper background wells for each group and inactivate the background wells of other groups. Fi...

Watch this Scientific Journal Video about Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells at JoVE.com

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

In this work, we describe a modified protocol to test mitochondrial respiratory substrate flux using recombinant perfringolysin O in combination with microplate-based respirometry. With this protocol, we show how metformin affects mitochondrial respiration of two different tumor cell lines.

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or ...

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Research

JoVE Journal

Biology

2.4K Views.  Charles University. Omdat het mitochondriale substraatflux test die kan worden beïnvloed door verschillende pathologieën of behandelingen. Vandaag zullen we het bijvoorbeeld gebruiken om de reactie van kankercellen op de behandeling met metformine te onthullen. Het vereist een minimaal aantal cellen, terwijl het voldoende replicaties en passende controle voor elk afzonderlijk materiaal heeft.De analyzer is alleen voor onderzoeksdoeleinden. Deze techniek kan fouten in het mitochondriale metabolisme ident...

Video: Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Developmental Biology

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.

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 ...

Biochemistry

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

Cancer Research

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Mitochondrial Functional Assessment in Brush Lizard Liver Mitochondria

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

Immunology and Infection

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to investigate metabolic capability. To obtain the measures of mitochondrial respiration, fresh biological samples must be used, and the entire laboratory procedure must be completed within approximately 2 h. Furthermore, multiple pieces of specialized equipment are required to perform these laboratory assays. This creates a challenge for measuring mitochondrial respiration in the tissues of wild animals living far from physiology laboratories as live tissue cannot be preserved for very long after collection in the field. Moreover, transporting live animals over long distances induces stress, which can alter mitochondrial energetics.
This manuscript introduces the Auburn University (AU) MitoMobile, a mobile mitochondrial physiology laboratory that can be taken into the field and used on-site to measure mitochondrial metabolism in tissues collected from wild animals. The basic features of the mobile laboratory and the step-by-step methods for measuring isolated mitochondrial respiration rates are presented. Additionally, the data presented validate the success of outfitting the mobile mitochondrial physiology laboratory and making mitochondrial respiration measurements. The novelty of the mobile laboratory lies in the ability to drive to the field and perform mitochondrial measurements on the tissues of animals captured on site.

We designed and constructed a mobile laboratory to measure respiration rates in isolated mitochondria of wild animals captured at field locations. Here, we describe the design and outfitting of a mobile mitochondrial laboratory and the associated laboratory protocols.

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to ...

High-Resolution Analysis of Mitochondrial Function in Human PBMCs

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes&#160;do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

Author Spotlight: New Insights into PBMC Mitochondrial Responses Using Fluorespirometry

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

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