The figures produced in this manuscript were created with BioRender.com. We are grateful to Amy Walker for providing feedback on this article. J.B.S. was supported by the Worcester Foundation for Biomedical Research Grant.

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

<ol>
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The authors have no conflicts of interest to report.

As emerging research demonstrates that mammalian mitochondria can function without consuming molecular oxygen, it is of the utmost importance for researchers to employ orthogonal assays, beyond OCR measurements, to accurately quantify mitochondrial function. Here, we compiled a series of assays that can be used to directly assess the activities of complex I, complex II, complex V, and DHODH by measuring the mitochondrial NAD+/NADH balance, the utilization of adaptive terminal electron acceptors, the production of ATP, de novo pyrimidine biosynthesis, and mitochondrial-derived ROS. Notably, these assays more directly measure mitochondrial function than OCR measurements. Furthermore, these assays provide researchers with tractable ways to quantify mitochondrial function during hypoxia, for which OCR measurements are largely irrelevant due to fumarate being used as the favored terminal electron acceptor. Finally, the proliferation-based methods described here are more cost-effective than classical respirometry experiments, thus providing a broadly accessible way to study mitochondrial function in mammalian systems.
There are key considerations when utilizing these assays to measure mitochondrial function in cultured cells. Regarding the proliferation assays, it is important to adjust the number of cells seeded for the doubling rate of each cell line. The cells should be seeded to at least 10% confluence and with enough space to allow for three to four doublings so that differences in proliferation can be quantified. Another consideration for each assay is the concentration of the small molecules used as controls for the activities of each ETC complex. As different cell lines may exhibit different sensitivities to these inhibitors, it is critical to test the dose of these small molecules to identify the optimal concentration.
A universal limitation of assays studying mitochondrial function in vitro, including OCR measurements and all the assays described here, is the metabolic composition of the culture medium. Standard cell culture medium tends to bias systems into superficially high levels of mitochondrial function. For example, supraphysiological glutamine levels increase its anaplerosis of the TCA cycle25, which fuels mitochondrial NADH synthesis and, consequently, increases oxidative phosphorylation. Similarly, the partial pressure of oxygen ranges between 3 mmHg and 100 mmHg (approximately 0.1%-13% O2) in mammalian tissues but is atmospheric (140 mmHg, approximately 21%) in vitro26,27. This excess O2 maximizes the mitochondrial respiratory capacity and superoxide production28. Recently, efforts have been made to design culture media to be more physiological29,30. Notably, culturing cells in human plasma-like media decreases mitochondrial respiration in some cancer cell lines30, mitochondrial ROS in T cells31, and mitochondrial adaptations to cancer therapeutics32. Thus, it is critical to be mindful of the composition of the culture media being used and understand how it may impact the mitochondrial function.
Another important and universal limitation in the interpretation of mitochondrial function is the potential for differences in the number of mitochondria. It is, therefore, critical to measure the mitochondrial content through either the quantification of mtDNA33, the measurement of mitochondrial mass with membrane potential-insensitive dyes34, or western blotting of mitochondrial markers. This is a critical control so that a decrease in the number of mitochondria is not mistaken for a decrease in mitochondrial function.
There are also specific limitations and troubleshooting that apply to the assays described here. First, given that differentiated cells do not proliferate, the proliferation-based assays will not be useful for assessing mitochondrial function in this context. A key limitation of the 13C4-aspartate tracing protocol to measure DHODH activity is that aspartate uptake in cells can be extremely inefficient35. To overcome this potential limitation, researchers can overexpress the aspartate transporter, SLC1A3, to facilitate 13C4-aspartate uptake35.
A limitation of the protocol using 13C5-glutamine tracing to measure SDH activity is that this assay requires cells to utilize the reductive carboxylation pathway to enrich the M+3 isotopologues in order to measure the reverse activity. Some cell lines are incapable of reductive carboxylation flux due to low ATP citrate lyase expression36, insufficient HIF stabilization37, or an &#945;-KG:citrate ratio that is too low38. To overcome this limitation, one could utilize 13C4-aspartate tracing to measure the SDH forward and reverse activities7. In this assay, the SDH forward activity can be measured by the ratio of fumarate M+2:succinate M+2 and the reverse reaction by succinate M+4:fumarate M+4. Notably, this tracing circumvents most of the enzymes in the reductive carboxylation pathway.
A limitation of the complex I activity assay using DCPIP reduction as the readout is that the mitochondria are not structurally intact. The process of freeze-thawing the mitochondria to enable their NADH uptake for the assay can certainly tarnish the structural integrity of the mitochondrial membrane39. This assay should be performed in parallel with assays such as the complex I proliferation assay to ensure that the changes in complex I activity observed are also true with intact&#160;cells.
In future studies, some of these techniques can be adapted for measuring mitochondrial functions in vivo using model organisms such as mice and Caenorhabditis elegans. The current methods used to measure mitochondrial function in vivo are centered on the organismal-level OCR, specifically the respiratory exchange rate when using mouse models. A clear limitation of this method is that oxygen serves many biochemical and signaling functions beyond its role as a ubiquitous terminal electron acceptor in the mitochondrial ETC. For example, oxygen is &#34;consumed&#34; by the catalytic activity of enzymes in the dioxygenase family. Although these enzymes contribute to the cellular oxygen consumption rate, they do not participate in, regulate, or reflect mitochondrial function. Classical respirometry experiments in vitro typically control for &#34;non-mitochondrial OCR&#34;, whereas organismal respiratory exchange ratio (RER) experiments cannot control for this, limiting the interpretation of RER as a metric for mitochondrial function in vivo. However, it is feasible to adapt the protocols to measure DHODH activity via&#160;13C4-aspartate tracing, complex II activity via&#160;13C5-glutamine tracing, complex I activity on mitochondria purified from tissues, and mitochondrial ROS using LC-MS friendly compounds such as MitoB in order to measure mitochondrial function in vivo. These direct assays to interrogate mitochondrial functions, in combination with classical respirometry experiments, provide researchers with a more comprehensive and accurate assessment of mitochondrial function in mammalian cells and tissues.

Mitochondrial function is a critical metric of cellular health, as it sustains key biosynthetic, bioenergetic, and signaling functions in mammalian cells1. The vast majority of mitochondrial functions require electron flow through the electron transport chain (ETC), and disruptions in electron flow in the ETC cause severe mitochondrial disease2. The ETC is comprised of a series of reduction and oxidation (redox) reactions that are embedded in the inner mitochondrial membrane, and these electron transfer reactions release free energy that can be harnessed to support ATP synthesis, physiological processes such as thermogenesis, biosynthetic pathways such as de novo pyrimidine biosynthesis, and the balance of the redox status of co-factors such as NADH. ETC complex I and&#160;III produce reactive oxygen species (ROS)3,4,5, which, in turn, regulate signaling key pathways such as HIF, PI3K, NRF2, NF&#954;B, and MAPK6. Consequently, metrics of electron flow in the ETC are classically used as a proxy for mitochondrial function in mammalian cells.
Respirometry experiments are frequently employed to measure mitochondrial function in mammalian cells. Since O2 is the most ubiquitous terminal electron acceptor for the mammalian ETC, its reduction is used as a proxy for mitochondrial function. However, emerging evidence demonstrates that mammalian mitochondria can employ fumarate as an electron acceptor to sustain mitochondrial functions that depend on the ETC, including de novo pyrimidine biosynthesis7, NADH oxidation7, and the detoxification of hydrogen sulfide8. Thus, in certain contexts, especially in hypoxic environments, measurements of the O2 consumption rate (OCR) do not provide a precise or accurate indication of mitochondrial function.
Here, we outline a series of assays that can be employed to measure mitochondrial function independently of the OCR. We provide assays to directly measure complex I-mediated NADH oxidation, dihydroorotate dehydrogenase-mediated de novo pyrimidine biosynthesis, complex V-dependent ATP synthesis, the net directionality of the succinate dehydrogenase (SDH) complex, and mitochondrial-derived ROS. These assays are meant to be performed on cultured mammalian cells, although many can be adapted to study mitochondrial functions in vivo. Notably, the assays described in this protocol are more direct measurements of mitochondrial functions than the OCR. Moreover, they enable the measurement of mitochondrial function in hypoxia, a context in which the OCR is not an indicative measurement. Taken together, these assays, in combination with classical respirometry experiments, will provide researchers with a more comprehensive assessment of mitochondrial function in mammalian cells.

<table><tbody><tr><td>&amp;nbsp;1.5 mL tube</td><td>Cell Treat</td><td>667443</td><td></td></tr><tr><td>2.0 mL tube</td><td>Cell Treat</td><td>229446</td><td></td></tr><tr><td>6-well plate</td><td>Cell Treat</td><td>229106</td><td></td></tr><tr><td>12-well plate</td><td>Cell Treat</td><td>229112</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;4&lt;/sub&gt;-aspartate</td><td>Sigma-Aldrich</td><td>604852</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;5&lt;/sub&gt;-Glutamine</td><td>Cambridge Isotope Laboratories</td><td>285978-14-5</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>Cell Treat</td><td>667411</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Cell Treat</td><td>667421</td><td></td></tr><tr><td>150 mm tissue culture dish</td><td>Cell Treat</td><td>229651</td><td></td></tr><tr><td>1x Phosphate-buffered saline</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>2,6-dichlorophenolindophenol</td><td>Honeywell</td><td>33125</td><td></td></tr><tr><td>Ammonium Carbonate</td><td>Sigma-Aldrich</td><td>37999</td><td></td></tr><tr><td>Antimycin</td><td>Sigma-Aldrich</td><td>A8674</td><td></td></tr><tr><td>Ascentis Express C18</td><td>Sigma-Aldrich</td><td>53825-U</td><td></td></tr><tr><td>Bottle top filter 500 mL, 0.22 &amp;micro;m, PES 9 9 mm membrane diameter</td><td>Cell Treat</td><td>229717</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A3294</td><td></td></tr><tr><td>Brequinar</td><td>Sigma-Aldrich</td><td>SML0113</td><td></td></tr><tr><td>Cell Lifter, Double End Flat and Narrow Blade</td><td>Cell Treat</td><td>229305</td><td></td></tr><tr><td>CentriVap -105 Cold Trap</td><td>Labconco</td><td>7385020</td><td></td></tr><tr><td>Complete Protease Inhibitor Tablets</td><td>Sigma-Aldrich&amp;nbsp;</td><td>4693116001</td><td></td></tr><tr><td>Coulter Counter Cups</td><td>Fisher Scientific</td><td>07-000-694</td><td></td></tr><tr><td>Decylubiquinone</td><td>Sigma-Aldrich</td><td>D7911</td><td></td></tr><tr><td>DMSO</td><td>Invitrogen</td><td>D12345</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Eppendorf Centrifuge 5425R</td><td>Eppendorf</td><td>2231000908</td><td></td></tr><tr><td>Eppendorf Centrifuge 5910 Ri</td><td>Eppendorf</td><td>5943000343</td><td></td></tr><tr><td>Galactose</td><td>Sigma-Aldrich</td><td>G5388</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Glucose-free DMEM</td><td>Gibco</td><td>11966025</td><td></td></tr><tr><td>Glutamine-free DMEM</td><td>Thermo Fisher</td><td>11960044</td><td></td></tr><tr><td>Heat-Inactivated Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F4135</td><td></td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>HPLC-grade 35% Ammonium hydroxide</td><td>Thermo Scientific</td><td>460801000</td><td></td></tr><tr><td>HPLC-grade Acetonitrile</td><td>Sigma-Aldrich</td><td>900667</td><td></td></tr><tr><td>HPLC-grade Chloroform</td><td>Sigma-Aldrich</td><td>366927</td><td></td></tr><tr><td>HPLC-grade formic acid</td><td>Thermo Scientific</td><td>28905</td><td></td></tr><tr><td>HPLC-grade Isopropanol</td><td>Sigma-Aldrich</td><td>563935</td><td></td></tr><tr><td>HPLC-grade MeOH</td><td>Sigma-Aldrich</td><td>900688</td><td></td></tr><tr><td>HPLC-grade Water</td><td>Sigma-Aldrich</td><td>270733</td><td></td></tr><tr><td>Human Osteosarcome Cell Line 143B</td><td>ATCC</td><td>CRL-8303</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Sigma-Aldrich</td><td>320331-500ML</td><td></td></tr><tr><td>Isotone buffer</td><td>Beckman Coulter</td><td>8546719</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P2222</td><td></td></tr><tr><td>Mannitol</td><td>Sigma-Aldrich</td><td>M4125</td><td></td></tr><tr><td>MitoSox Red</td><td>Invitrogen</td><td>M36008</td><td></td></tr><tr><td>N-acetyl-L-cysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>Oligomycin</td><td>Sigma-Aldrich</td><td>75351-5MG</td><td></td></tr><tr><td>Pencillin Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Potter-Elvehjem Tissue Grinder, Size 21</td><td>Kimble</td><td>885502-0021</td><td></td></tr><tr><td>Pyruvate</td><td>Sigma-Aldrich</td><td>P5280</td><td></td></tr><tr><td>Pyruvate-free DMEM media</td><td>Gibco</td><td>11965175</td><td></td></tr><tr><td>Q Exactive Plus Mass Spectrometer</td><td>Thermo Scientific</td><td>726030</td><td></td></tr><tr><td>ReCO&lt;sub&gt;2&lt;/sub&gt;ver Incubator</td><td>Baker</td><td></td><td></td></tr><tr><td>Refrigerated Centrivap Benchtop Vacuum Concentrator</td><td>Labconco</td><td>7310020</td><td></td></tr><tr><td>RIPA Buffer</td><td>Millipore Sigma</td><td>20188</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>SeQuant ZIC-pHILIC 5&amp;mu;m 150 x 2.1 mm analytical column</td><td>Sigma-Aldrich</td><td>1.50460.0001</td><td></td></tr><tr><td>SeQuant ZIC-pHILIC guard kit</td><td>Millipore Sigma</td><td>1.50438.0001</td><td></td></tr><tr><td>Sodium Hydroxide, Pellets</td><td>Millipore Sigma</td><td>567530-250GM</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>SW, TRACEFINDER 5.1 SP3</td><td>Thermo Scientific</td><td>OPTON-31001</td><td></td></tr><tr><td>Tert-butyl hydroperoxide solution</td><td>Sigma-Aldrich</td><td>458139</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>93352</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Gibco</td><td>25-200-114</td><td></td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3003</td><td></td></tr><tr><td>VANQUISH HORIZON / FLEX HPLC</td><td>Thermo Scientific</td><td>VF-S01-A-02</td><td></td></tr><tr><td>Z2 Coulter Particle count and size analyzer</td><td>Beckman Coulter</td><td>BZ10131270</td><td></td></tr></tbody></table>

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.

The activities of DHODH, complex I, and complex V can all be assessed using proliferation assays. Upon deprivation of uridine from the culture medium, the cells become more dependent on the de novo pathway for pyrimidine biosynthesis. Thus, when cells were challenged to proliferate in a uridine-free medium, they were more sensitive to the inhibition of DHODH activity by brequinar than cells cultured in a medium containing uridine (Figure 6A). Similarly, the deprivation of pyruvate from the culture medium renders the cells more dependent on complex I activity for proliferation. Thus, when cells were challenged to proliferate in a pyruvate-free medium, they were more sensitive to the inhibition of complex I activity by rotenone than cells cultured in a pyruvate-containing medium (Figure 6B). Complex V activity can be assessed by challenging cells to proliferate in a medium containing galactose instead of glucose. As galactose yields net zero ATP in glycolysis, cells growing in this fuel are more reliant on mitochondrial ATP synthesis via complex V activity. Thus, cells proliferating in a galactose-containing medium were more sensitive to complex V inhibition by oligomycin than cells proliferating in a glucose-containing medium (Figure 6C).
SDH activity can be measured using 13C5-glutamine tracing and by monitoring its incorporation into fumarate and succinate isotopologues. In vehicle-treated conditions, the SDH complex favored the forward activity, and the incorporation of 13C4-succinate into 13C4-fumarate was higher than the incorporation of 13C3-fumarate into 13C3-succinate (Figure 7). In antimycin-treated conditions, the SDH complex favored the reverse activity, and the incorporation of 13C3-fumarate into 13C3-succinate was greater than the incorporation of 13C4-succinate into 13C4-fumarate (Figure 7).
Superoxide production inside the mitochondria can be measured using the fluorescent reporter MitoSox, which generates 2-hydroxy-mitoethidium upon reaction with superoxide. In this study, cells treated with MitoSox in the presence of tertbutyl hydrogen peroxide had higher levels of 2-hydroxy-mitoethidium in a manner that was suppressed by the addition of NAC, an antioxidant that quenches cellular ROS (Figure 8).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65184/65184fig01.jpg" /> 
Figure 1: Mechanistic basis for the complex V proliferation assay. Oxidation of glucose and galactose via glycolysis. Glucose yields net two ATP from glycolysis, whereas galactose yields net zero ATP because UTP synthesis is required for UDP-galactose. Thus, cells grown in galactose are more dependent on mitochondrial ATP synthesis due to a lack of ATP produced from glycolysis. Abbreviations: GALK = galactokinase; GALT = galactose-1-phosphate uridylyltransferase; PGM1 = phosphoglucomutase 1; GPI = glucose-6-phosphate isomerase, UGP = UDP-glucose pyrophosphorylase; GALE = UDP-galactose-4-epimerase; NDK = nucleotide diphosphate kinase; UMPK = uridine monophosphate kinase; HK = hexokinase; PFK = phosphofructokinase; ALDO = aldolase; TPI = triosephosphate isomerase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; PGK = phosphoglycerate kinase; PGM = phosphoglucomutase; ENO = enolase; PK = pyruvate kinase. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65184/65184fig02.jpg" /> 
Figure 2: Mechanistic basis for the complex I proliferation assay. Schematic of the metabolic pathways that are altered upon the inhibition of complex I activity. In high-pyruvate media, complex I inhibition is bypassed via LDH-mediated NADH oxidation. In low-pyruvate media, this adaptation is less feasible, making cells more reliant on complex I activity to reoxidize NADH. Abbreviations: LDH = lactate dehydrogenase; TCA cycle = tricarboxylic acid cycle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65184/65184fig03.jpg" /> 
Figure 3: Schematic of the DHODH reaction upon 13C4-aspartate tracing. Brequinar inhibits dihydroorotate oxidation to orotate, thus preventing the downstream synthesis of UMP. 13C4-aspartate is incorporated into 13C3-UMP via DHODH activity. Abbreviations: OMM = outer mitochondrial membrane; IMM = inner mitochondrial membrane; DHODH = dihydroorotate dehydrogenase. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65184/65184fig04.jpg" /> 
Figure 4: Measuring forward and reverse complex II activity via&#160;13C5-glutamine tracing. To measure forward complex II activity (left), the incorporation of 13C5-glutamine into 13C4-succinate and 13C4-fumarate is monitored. To measure reverse complex II activity (right), the incorporation of 13C5-glutamine into 13C3-succinate and 13C3-fumarate is monitored. Abbreviations: SDH = succinate dehydrogenase; CytC = cytochrome C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65184/65184fig05.jpg" /> 
Figure 5: MitoSox Red reaction with superoxide. The reaction of MitoSox with mitochondrial superoxides to form 2-OH-MitoE2+. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65184/65184fig06.jpg" /> 
Figure 6: Proliferation-based assays to measure mitochondrial function (A) Proliferation of 143B osteosarcoma cells treated with 5 uM brequinar, a DHODH inhibitor, in medium with &#177;100 ug/mL uridine. Data are mean &#177; SEM; N = 3 per condition. (B) Proliferation of 143B osteosarcoma cells treated with the 2 uM rotenone, a complex I inhibitor, in medium with &#177;5 mM pyruvate. Data are mean &#177; SEM; N = 3 per condition. (C) Proliferation of 143B osteosarcoma cells treated with the 5 uM oligomycin, a complex V inhibitor, in medium with either 10 mM glucose or 10 mM galactose as the sole central carbon source. Data are mean &#177; SEM; N = 3 per condition. * indicates p &#60; 0.05 using a one-way ANOVA test in Graphpad Prism. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65184/65184fig07.jpg" /> 
Figure 7: 13C5-glutamine tracing to measure complex II activity. Succinate oxidation and fumarate reduction (SDH reverse) in DMSO and 500 nM antimycin A-treated Caki1 and DLD1 cells. Data represent mean &#177; SEM; N = 3 per condition. *** indicates p &#60; 0.05 using an unpaired t-test in GraphPad Prism. Abbreviations: SDH = succinate oxidation; SDH reverse = fumarate reduction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65184/65184fig08v2.jpg" /> 
Figure 8: LCMS-based MitoSox assay to detect superoxide. Extracted ion chromatogram of 2-OH-Mito E2+ isolated from Caki1 cells treated with MitoSox for 30 min in the presence of tBuOOH &#177; NAC. Abbreviations: LCMS = liquid chromatography-mass spectrometry; NAC = N-acetyl cysteine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of reagents, buffers, and media used in this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184_Table 1 Reaction Compositions_Revision (2).xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals at JoVE.com

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.

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 ...

oxygen-independent-assays-to-measure-mitochondrial-function-in-mammals

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.5K Views.  University of Massachusetts Chan Medical School. Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

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

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 ...

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we&#160;outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.

We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response.

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

Cancer Research

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 ...

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

Author Spotlight: Uncovering the Role of Mitochondrial Calcium Phosphate in Heart Failure and Bioenergetics

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

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 ...

Biology

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

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.

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 ...

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.