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S-1 MitoPlates™ from Biolog enable the characterization of mitochondria's function in live cells by measuring the rates of electron flow into and through the electron transport chain from different NADH or FADH 2 producing metabolic substrates. This technology uses 96-well microplates pre-coated with triplicate repeats of a set of 31 substrates. Those 31 metabolic substrates have different routes of entry into the mitochondria, use different transporters, and are also oxidated by different dehydrogenases, producing reducing equivalents in the form of NADH or FADH 2 . The electrons produced upon oxidation of NADH or FADH 2 at complex I or II, respectively, then travel to cytochrome c, where a tetrazolium redox dye (MC) can act as terminal acceptor, turning purple and absorbing at 590 nm. This mechanism allows the evaluation of cellular substrate preference by following the kinetics of MC reduction in the presence of selected substrates.In this chapter, we describe the step-by-step protocol to prepare an experiment using MitoPlate S-1 array and the OmniLog instrument to assess the metabolism of human dermal fibroblasts. We also give detailed information on how to analyze the raw data generated by the Biolog Data Analysis software to extract meaningful information and produce useful data visualizations, using reproducible methods based on a single structured dataset., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In vitro and ex vivo studies are crucial for mitochondrial research, offering valuable insights into cellular mechanisms and aiding in diagnostic and therapeutic strategies. Accurate in vitro models rely on adequate cell culture conditions, such as the composition of culture media and oxygenation levels. These conditions can influence energy metabolism and mitochondrial activities, thus impacting studies involving mitochondrial components, such as the effectiveness of anticancer drugs. This chapter focuses on practical guidance for creating setups that replicate in vivo microenvironments, capturing the original metabolic context of cells. We explore protocols to better mimic the physiological cell environment, promote cellular reconfiguration, and prime cells according to the modeled context. The first part is dedicated to the use of human dermal fibroblasts, which are a promising model for pre-clinical mitochondrial research due to their adaptability and relevance to human mitochondrial physiology. We present an optimized protocol for gradually adjusting extracellular glucose levels, which demonstrated significant mitochondrial, metabolic, and redox remodeling in normal adult dermal fibroblasts. The second part is dedicated to replication of tumor microenvironments, which are relevant for studies targeting cellular energy metabolism to inhibit tumor growth. Currently available physiological media can mimic blood plasma metabolome but not the specific tumor microenvironment. To address this, we describe optimized media formulation and oxygenation protocols, which can simulate the tumor microenvironment in cell culture experiments. Replicating in vivo microenvironments in in vitro and ex vivo studies can enhance our understanding of cellular processes, facilitate drug development, and advance personalized therapeutics in mitochondrial medicine., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with a rapid progression and no effective treatment. Metabolic and mitochondrial alterations in peripheral tissues of ALS patients may present diagnostic and therapeutic interest. We aimed to identify mitochondrial fingerprints in lymphoblast from ALS patients harboring SOD1 mutations (mutSOD1) or with unidentified mutations (undSOD1), compared with age-/sex-matched controls. Three groups of lymphoblasts, from mutSOD1 or undSOD1 ALS patients and age-/sex-matched controls, were obtained from Coriell Biobank and divided into 3 age-/sex-matched cohorts. Mitochondria-associated metabolic pathways were analyzed using Seahorse MitoStress and ATP Rate assays, complemented with metabolic phenotype microarrays, metabolite levels, gene expression, and protein expression and activity. Pooled (all cohorts) and paired (intra-cohort) analyses were performed by using bioinformatic tools, and the features with higher information gain values were selected and used for principal component analysis and Naïve Bayes classification. Considering the group as a target, the features that contributed to better segregation of control, undSOD1, and mutSOD1 were found to be the protein levels of Tfam and glycolytic ATP production rate. Metabolic phenotypic profiles in lymphoblasts from ALS patients with mutSOD1 and undSOD1 revealed unique age-dependent different substrate oxidation profiles. For most parameters, different patterns of variation in experimental endpoints in lymphoblasts were found between cohorts, which may be due to the age or sex of the donor. In the present work, we investigated several metabolic and mitochondrial hallmarks in lymphoblasts from each donor, and although a high heterogeneity of results was found, we identified specific metabolic and mitochondrial fingerprints, especially protein levels of Tfam and glycolytic ATP production rate, that may have a diagnostic and therapeutic interest., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mitochondria play a key role in cell death and its regulation. The permeabilization of the outer mitochondrial membrane, which is mainly controlled by proteins of the BCL-2 family, is a key event that can be directly induced by different signaling pathways, including p53-mediated, and results in the release of proapoptotic factors to the cytosol, such as cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor-of-apoptosis (IAP) binding protein with low pI (SMAC/Diablo), Omi serine protease (Omi/HtrA2), apoptosis-inducing factor (AIF), or endonuclease G (Endo-G). Hence, the determination of subcellular localization of these proteins is extremely important to predict cell fate and elucidate the specific mechanism of apoptosis. Here we describe experimental protocols that can be used to study the subcellular location of different proapoptotic proteins to be used in basic cell biology and toxicology studies.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the huntingtin protein. Mitochondrial dysfunction associated with energy failure plays an important role in this untreated pathology. In the present work, we used lymphoblasts obtained from HD patients or unaffected parentally related individuals to study the protective role of insulin-like growth factor 1 (IGF-1) versus insulin (at low nM) on signaling and metabolic and mitochondrial functions. Deregulation of intracellular signaling pathways linked to activation of insulin and IGF-1 receptors (IR,IGF-1R), Akt, and ERK was largely restored by IGF-1 and, at a less extent, by insulin in HD human lymphoblasts. Importantly, both neurotrophic factors stimulated huntingtin phosphorylation at Ser421 in HD cells. IGF-1 and insulin also rescued energy levels in HD peripheral cells, as evaluated by increased ATP and phosphocreatine, and decreased lactate levels. Moreover, IGF-1 effectively ameliorated O2 consumption and mitochondrial membrane potential (Δψm) in HD lymphoblasts, which occurred concomitantly with increased levels of cytochrome c. Indeed, constitutive phosphorylation of huntingtin was able to restore the Δψm in lymphoblasts expressing an abnormal expansion of polyglutamines. HD lymphoblasts further exhibited increased intracellular Ca(2+) levels before and after exposure to hydrogen peroxide (H2O2), and decreased mitochondrial Ca(2+) accumulation, being the later recovered by IGF-1 and insulin in HD lymphoblasts pre-exposed to H2O2. In summary, the data support an important role for IR/IGF-1R mediated activation of signaling pathways and improved mitochondrial and metabolic function in HD human lymphoblasts.

Mitochondria play a key role in cell death and its regulation. The permeabilization of the outer mitochondrial membrane which is mainly controlled by proteins of the BCL-2 family, is a key event that can be directly induced by p53 and results in the release of pro-apoptotic factors to the cytosol, such as cytochrome c, second mitochondria derived activator of caspases/direct inhibitor-of-apoptosis (IAP) binding protein with low pI (SMAC/Diablo), Omi serine protease (Omi/HtrA2), apoptosis inducing factor (AIF), or endonuclease G (Endo-G). Hence, the determination of subcellular localization of these proteins is extremely important to predict cell fate and elucidate the specific mechanism of apoptosis. Here we describe the procedures that can be used to study the subcellular location of different pro-apoptotic proteins to be used in basic cell biology and toxicology studies.