Name: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals (Video) | JoVE
Uploaded: 2023-05-19T12:40:00
Duration: 1 min 14 s
Description: Watch this Scientific Journal Video about Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals at JoVE.com

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>
	<li>Spinelli, J. B., Haigis, M. 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+multifaceted+contributions+of+mitochondria+to+cellular+metabolism.">The multifaceted contributions of mitochondria to cellular metabolism.</a> Nature Cell Biology. 20 (7), 745-754 (2018).</li><li>Gorman, G. 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=Mitochondrial+diseases.">Mitochondrial diseases.</a> Nature Reviews Disease Primers. 2, 16080 (2016).</li><li>Chandel, N. 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=Reactive+oxygen+species+generated+at+mitochondrial+complex+III+stabilize+hypoxia-inducible+factor-1alpha+during+hypoxia:+A+mechanism+of+O2+sensing.">Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: A mechanism of O2 sensing.</a> Journal of Biological Chemistry. 275 (33), 25130-25138 (2000).</li><li>Cadenas, E., Boveris, A., Ragan, C. I., Stoppani, A. O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+superoxide+radicals+and+hydrogen+peroxide+by+NADH-ubiquinone+reductase+and+ubiquinol-cytochrome+C+reductase+from+beef-heart+mitochondria.">Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome C reductase from beef-heart mitochondria.</a> Archives of Biochemistry and Biophysics. 180 (2), 248-257 (1977).</li><li>Murphy, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+mitochondria+produce+reactive+oxygen+species.">How mitochondria produce reactive oxygen species.</a> Biochemical Journal. 417 (1), 1-13 (2009).</li><li>Schieber, M., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+function+in+redox+signaling+and+oxidative+stress.">ROS function in redox signaling and oxidative stress.</a> Current Biology. 24 (10), 453-462 (2014).</li><li>Spinelli, J. B., 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=Fumarate+is+a+terminal+electron+acceptor+in+the+mammalian+electron+transport+chain.">Fumarate is a terminal electron acceptor in the mammalian electron transport chain.</a> Science. 374 (6572), 1227-1237 (2021).</li><li>Kumar, R., 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=A+redox+cycle+with+complex+II+prioritizes+sulfide+quinone+oxidoreductase-dependent+H(2)S+oxidation.">A redox cycle with complex II prioritizes sulfide quinone oxidoreductase-dependent H(2)S oxidation.</a> Journal of Biological Chemistry. 298 (1), 101435 (2022).</li><li>Warburg, O., Geissler, A. W., Lorenz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+growth+of+cancer+cells+in+media+in+which+glucose+is+replaced+by+galactose.">On growth of cancer cells in media in which glucose is replaced by galactose.</a> Hoppe-Seyler's Zeitschrift f&#252;r Physiologische Chemie. 348 (12), 1686-1687 (1967).</li><li>Marroquin, L. D., Hynes, J., Dykens, J. A., Jamieson, J. D., Will, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circumventing+the+Crabtree+effect:+replacing+media+glucose+with+galactose+increases+susceptibility+of+HepG2+cells+to+mitochondrial+toxicants.">Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants.</a> Toxicological Sciences. 97 (2), 539-547 (2007).</li><li>Attardi, G., King, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cells+lacking+mtDNA:+Repopulation+with+exogenous+mitochondria+by+complementation.">Human cells lacking mtDNA: Repopulation with exogenous mitochondria by complementation.</a> Science. 246 (4929), 500-503 (1989).</li><li>Bodnar, A. G., Cooper, M., Leonard, J. V., Schapira, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory-deficient+human+fibroblasts+exhibiting+defective+mitochondrial+DNA+replication.">Respiratory-deficient human fibroblasts exhibiting defective mitochondrial DNA replication.</a> Biochemical Journal. 305, 817-822 (1995).</li><li>Gregoire, M., Morais, R., Quilliam, M. A., Gravel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+auxotrophy+for+pyrimidines+of+respiration-deficient+chick+embryo+cells.">On auxotrophy for pyrimidines of respiration-deficient chick embryo cells.</a> European Journal of Biochemistry. 142 (1), 49-55 (1984).</li><li>Mackay, G. M., Zheng, L., vanden Broek, N. J., Gottlieb, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+metabolism+using+LC-MS+and+isotope+tracers.">Analysis of cell metabolism using LC-MS and isotope tracers.</a> Methods in Enzymology. 561, 171-196 (2015).</li><li>Heinrich, 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=Correcting+for+natural+isotope+abundance+and+tracer+impurity+in+MS-,+MS/MS-+and+high-resolution-multiple-tracer-data+from+stable+isotope+labeling+experiments+with+IsoCorrectoR.">Correcting for natural isotope abundance and tracer impurity in MS-, MS/MS- and high-resolution-multiple-tracer-data from stable isotope labeling experiments with IsoCorrectoR.</a> Scientific Reports. 8 (1), 17910 (2018).</li><li>Lee, P., Chandel, N. S., Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+adaptation+to+hypoxia+through+hypoxia+inducible+factors+and+beyond.">Cellular adaptation to hypoxia through hypoxia inducible factors and beyond.</a> Nature Reviews Molecular Cell Biology. 21 (5), 268-283 (2020).</li><li>Bisbach, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Succinate+can+shuttle+reducing+power+from+the+hypoxic+retina+to+the+O(2)-rich+pigment+epithelium.">Succinate can shuttle reducing power from the hypoxic retina to the O(2)-rich pigment epithelium.</a> Cell Reports. 31 (2), 107606 (2020).</li><li>Angebault, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idebenone+increases+mitochondrial+complex+I+activity+in+fibroblasts+from+LHON+patients+while+producing+contradictory+effects+on+respiration.">Idebenone increases mitochondrial complex I activity in fibroblasts from LHON patients while producing contradictory effects on respiration.</a> BMC Research Notes. 4, 557 (2011).</li><li>Liao, P. C., Bergamini, C., Fato, R., Pon, L. A., Pallotti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria+from+cells+and+tissues.">Isolation of mitochondria from cells and tissues.</a> Methods in Cell Biology. 155, 3-31 (2020).</li><li>Iwai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+accumulation+during+ischemia+induces+mitochondrial+damage+in+perfused+rat+hearts.">Sodium accumulation during ischemia induces mitochondrial damage in perfused rat hearts.</a> Cardiovascular Research. 55 (1), 141-149 (2002).</li><li>Murphy, E., Eisner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+intracellular+and+mitochondrial+sodium+in+health+and+disease.">Regulation of intracellular and mitochondrial sodium in health and disease.</a> Circulation Research. 104 (3), 292-303 (2009).</li><li>Lampl, T., Crum, J. A., Davis, T. A., Milligan, C., Del Gaizo Moore, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+analysis+of+mitochondria+from+cultured+cells+and+mouse+tissue.">Isolation and functional analysis of mitochondria from cultured cells and mouse tissue.</a> Journal of Visualized Experiments. (97), e52076 (2015).</li><li>Murphy, M. 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=Guidelines+for+measuring+reactive+oxygen+species+and+oxidative+damage+in+cells+and+in+vivo.">Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo.</a> Nature Metabolism. 4 (6), 651-662 (2022).</li><li>Xiao, Y., Meierhofer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+hydroethidine-based+probes+reliable+for+reactive+oxygen+species+detection.">Are hydroethidine-based probes reliable for reactive oxygen species detection.</a> Antioxidants and Redox Signaling. 31 (4), 359-367 (2019).</li><li>DeBerardinis, R. J., 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=Beyond+aerobic+glycolysis:+Transformed+cells+can+engage+in+glutamine+metabolism+that+exceeds+the+requirement+for+protein+and+nucleotide+synthesis.">Beyond aerobic glycolysis: Transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (49), 19345-19350 (2007).</li><li>Keeley, T. P., Mann, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+physiological+normoxia+for+improved+translation+of+cell+physiology+to+animal+models+and+humans.">Defining physiological normoxia for improved translation of cell physiology to animal models and humans.</a> Physiological Reviews. 99 (1), 161-234 (2019).</li><li>Ast, T., Mootha, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+and+mammalian+cell+culture:+Are+we+repeating+the+experiment+of+Dr.+Ox.">Oxygen and mammalian cell culture: Are we repeating the experiment of Dr. Ox.</a> Nature Metabolism. 1 (9), 858-860 (2019).</li><li>Gu, C., Jun, 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=Does+hypoxia+decrease+the+metabolic+rate.">Does hypoxia decrease the metabolic rate.</a> Frontiers in Endocrinology. 9, 668 (2018).</li><li>Voorde, J., 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=Improving+the+metabolic+fidelity+of+cancer+models+with+a+physiological+cell+culture+medium.">Improving the metabolic fidelity of cancer models with a physiological cell culture medium.</a> Scientific Advances. 5 (1), 7314 (2019).</li><li>Cantor, J. R., 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=Physiologic+medium+rewires+cellular+metabolism+and+reveals+uric+acid+as+an+endogenous+inhibitor+of+UMP+synthase.">Physiologic medium rewires cellular metabolism and reveals uric acid as an endogenous inhibitor of UMP synthase.</a> Cell. 169 (2), 258-272 (2017).</li><li>MacPherson, 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=Clinically+relevant+T+cell+expansion+media+activate+distinct+metabolic+programs+uncoupled+from+cellular+function.">Clinically relevant T cell expansion media activate distinct metabolic programs uncoupled from cellular function.</a> Molecular Therapy. Methods and Clinical Development. 24, 380-393 (2022).</li><li>Torres-Quesada, O., Doerrier, C., Strich, S., Gnaiger, E., Stefan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+cell+culture+media+tune+mitochondrial+bioenergetics+and+drug+sensitivity+in+cancer+cell+models.">Physiological cell culture media tune mitochondrial bioenergetics and drug sensitivity in cancer cell models.</a> Cancers. 14 (16), 3917 (2022).</li><li>Chan, S. W., Chen, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mtDNA+damage+using+a+supercoiling-sensitive+qPCR+approach.">Measuring mtDNA damage using a supercoiling-sensitive qPCR approach.</a> Methods in Molecular Biology. 554, 183-197 (2009).</li><li>Doherty, E., Perl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+mass+by+flow+cytometry+during+oxidative+stress.">Measurement of mitochondrial mass by flow cytometry during oxidative stress.</a> Reactive Oxygen Species. 4 (10), 275-283 (2017).</li><li>Birsoy, K., 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=An+essential+role+of+the+mitochondrial+electron+transport+chain+in+cell+proliferation+is+to+enable+aspartate+synthesis.">An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis.</a> Cell. 162 (3), 540-551 (2015).</li><li>Beigneux, A. 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=ATP-citrate+lyase+deficiency+in+the+mouse.">ATP-citrate lyase deficiency in the mouse.</a> Journal of Biological Chemistry. 279 (10), 9557-9564 (2004).</li><li>Gameiro, P. 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=In+vivo+HIF-mediated+reductive+carboxylation+is+regulated+by+citrate+levels+and+sensitizes+VHL-deficient+cells+to+glutamine+deprivation.">In vivo HIF-mediated reductive carboxylation is regulated by citrate levels and sensitizes VHL-deficient cells to glutamine deprivation.</a> Cell Metabolism. 17 (3), 372-385 (2013).</li><li>Fendt, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductive+glutamine+metabolism+is+a+function+of+the+alpha-ketoglutarate+to+citrate+ratio+in+cells.">Reductive glutamine metabolism is a function of the alpha-ketoglutarate to citrate ratio in cells.</a> Nature Communications. 4, 2236 (2013).</li><li>Lee, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+studies+of+isolated+mitochondria+from+normal+and+diseased+tissues.">Biochemical studies of isolated mitochondria from normal and diseased tissues.</a> Biochimica et Biophysica Acta. 1271 (1), 21-28 (1995).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;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>13C4-aspartate</td>
			<td>Sigma-Aldrich</td>
			<td>604852</td>
			<td></td>
		</tr>
		<tr>
			<td>13C5-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 &#181;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&#160;</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&#8217;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>K2HPO4</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>ReCO2ver 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&#956;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.

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

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/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/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/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/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/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/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/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/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/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

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals (Video) | JoVE 

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 ...