While we have reviewed evidence supporting CP as the bona fide demarcator of the maximal metabolic steady state, it is essential that great care is taken in its estimation (Mattioni Maturana etal. 2018; Muniz‐Pumares etal. 2019). There are two methods by which CP can be assessed: the ‘conventional’ approach in which CP is modeled from a series of severe‐intensity ‘prediction trials’ performed to the limit of tolerance at different speeds or power outputs (Monod and Scherrer 1965; Poole etal. 1988); and the 3‐min all‐out test in which, as the name implies, subjects exercise maximally for 3min with the end‐test power representing the CP and the total work done above CP representing the W′ (Burnley etal. 2006; Vanhatalo etal. 2007). If the CP is estimated using the conventional approach, important considerations include the number of trials and their duration (Hill 1993; Bishop etal. 1998; Triska etal. 2018). It is essential that subjects give their maximum effort in each trial and that cadence is consistent across all trials. Ideally the shortest trial should be 2–3min and the longest should be more than 10 but no longer than 15min (Hill 1993; Vanhatalo etal. 2011a). It has been recommended that there should be at least a 5min difference between the shortest and longest trials (Bishop etal. 1998) but the goodness of hyperbolic fit is improved by making the range of times to exhaustion as broad as possible (i.e., 8–12min) within the severe‐intensity domain. The precise duration of the prediction trials is of secondary importance to the attainment of V ˙O 2max, but it is unusual for V ˙O 2max to be attained if exercise duration is shorter than 1–2min or longer than 15–20min (Hill etal. 2002; Vanhatalo etal. 2016). V ˙O 2 should be measured during each trial to verify attainment of V ˙O 2max, with this typically defined as the end‐exercise V ˙O 2 exceeding 95% of the V ˙O 2max measured during ramp incremental exercise, to allow for biological and methodological day‐to‐day variability (Katch etal. 1982). Detmer, S. A. & Chan, D. C. Functions and dysfunctions of mitochondrial dynamics. Nat. Rev. Mol. Cell Biol. 8, 870 (2007). Scarpulla, R. C., Vega, R. B. & Kelly, D. P. Transcriptional integration of mitochondrial biogenesis. Trends Endocrinol. Metab. 23, 459–466 (2012). Allen, S. G. et al. Macrophages enhance migration in inflammatory breast cancer cells via RhoC GTPase signaling. Sci. Rep. 6, 39190 https://www.nature.com/articles/srep39190#supplementary-information (2016). A primary output of cellular metabolism is chemical energy in the form of adenosine triphosphate (ATP). The major bioenergetic pathways that generate ATP in a cell are glycolysis and mitochondrial respiration 1, 2. Production of ATP from the mitochondria is coupled to the generation of reducing power in the tricarboxylic acid (TCA) cycle, and the respiration-dependent formation of a proton gradient by the electron transport chain (ETC).

Despite the widespread use of the Seahorse Bioanalyzer technology, acquisition of reliable data requires effective normalization strategies to correct for cell density. Multiple normalization methodologies have been used with varying degrees of acceptance by the research community. Examples include normalization to post-assay protein harvest or post-assay cell counting, normalization to pre-assay cell counting 22, or normalization via one of a variety of chemical colorimetric or fluorometric readouts (e.g., MTT, ATPGlo, WST-1). Specifically in a recent study employing small-interfering RNA (siRNA)/short-hairpin RNA (shRNA) screening, Hoechst nuclei staining coupled with automated nuclei count was demonstrated to have better performance than other normalization methods 23. Indeed, this strategy has been recently incorporated into the Seahorse pipeline to more adequately control for cell number with the merger of BioTek Cytation5 and Seahorse XF assay platforms 24. Herein, we optimize and extend this previous work, as nuclei staining can also be applied to determine cell cycle distribution 25, 26; an important cellular characteristic that affects bioenergetics. Importantly, mitochondrial bioenergetics have been previously shown to coordinate with cell cycle dynamics 27, 28, further supporting the use of nuclei counterstaining in conjunction with the metabolic flux assay. Methodological aspects of maximal lactate steady state‐implications for performance testing. Eur. J. Appl. Physiol. 89:95–99. [ PubMed] [ Google Scholar] Wang, J.-B. et al. Targeting mitochondrial glutaminase activity inhibits oncogenic transformation. Cancer Cell 18, 207–219 (2010).While recent emerging technologies have permitted more precise examination of mitochondrial functions and properties, each of these techniques are typically performed independently 13, 14. This is problematic in the sense that mitochondria are rapidly undergoing (bio)chemical, morphological, physiochemical, thermodynamic, and other changes at any given time; making experiment-to-experiment comparisons challenging. Therefore, capturing bioenergetic and functional data in a single multifunctional assay has the potential to yield greater, more controlled, and more precise mitochondrial information. Here, we describe an integrated platform that utilizes bioenergetic profiling technology alongside imaging of mitochondrial functions and properties to obtain a richer data set from a single experiment. The potential for long-term risks to health far outweighs any health benefits or cost savings associated with consuming synthetic vitamins. Kuznetsov, A. V. et al. Mitochondrial ROS production under cellular stress: comparison of different detection methods. Anal. Bioanal. Chem. 400, 2383–2390 (2011).

The natural bioactive polyphenols of the spice turmeric called curcuminoids have been shown to counteract both inflammation and oxidative stress caused by exercise. Additional fluorescence-based dyes are similarly available to measure discrete mitochondrial parameters, including inner mitochondrial membrane potential (Δ ψ m) and mitochondrial reactive oxygen species (mtROS). Δ ψ m is generated by the proton pumping complexes of the ETC. The energy “stored” in the Δ ψ m is ultimately used to drive ATP production by complex V. While moderate fluctuations in the Δ ψ m can reflect normal functioning of mitochondria, sustained increases or drops can lead to mitochondrial pathology and/or target mitochondria for degradation. To build in the detection of Δ ψ m into our imaging platform, we utilized the fluorescent dye tetramethylrhodamine ethyl ester (TMRE). TMRE is sequestered in the matrix of active mitochondria based on the positive charge of the dye 33, 34, 35. Depletion of the Δ ψ m leads to loss of polarity and thus loss of dye sequestration and signal. Wynn, M. L. et al. RhoC GTPase is a potent regulator of glutamine metabolism and N-acetylaspartate production in inflammatory breast cancer cells. J. Biol. Chem. 291, 13715–13729 (2016). Department of Pathology and Institute of Gerontology, University of Michigan, Ann Arbor, MI, 48109, USATraba, J., Miozzo, P., Akkaya, B., Pierce, S. K. & Akkaya, M. An optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes. JoVE, e54918, https://doi.org/10.3791/54918 (2016). Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, Ann Arbor, MI, 48109, USA When combined with lifestyle efforts focusing on diet, exercise, sleep and stress, the combination of bergamot fruit and artichoke leaf extracts can provide significant health benefits. Crowley, L. C., Christensen, M. E. & Waterhouse, N. J. Measuring mitochondrial transmembrane potential by TMRE staining. Cold Spring Harb. Protocols 2016, https://doi.org/10.1101/pdb.prot087361 (2016). Reduce unhealthy fats from inside and outside the liver enabling it to process fat more effectively.