Notes

Sonographic look at baby nut sack, testes and epididymis.
We describe here reliable histochemical and immunohistochemical techniques to visualize mitochondria and respiratory chain dysfunction in tissue sections. These morphological methods have been widely used for years, and yet remain relevant to obtain insight into the pathogenesis of mitochondrial diseases. Today, mitochondrial medicine is changing rapidly and genetic information plays an increasing role in the diagnostic process, owing to advances in next-generation sequencing. However, tissue analysis and morphological categorization remain essential, especially when genetic abnormalities of unknown significance might complicate a diagnostic odyssey. Furthermore, tissue assessment is an essential step in laboratory investigation using animal or cell models, in order to assess the distribution, severity, and/or progression of the disease, and to evaluate the effects of possible treatments. Optimized and reproducible staining and imaging methodology are the key elements for accurate tissue assessment. When these methods are used properly and integrated with wisely chosen genetic and biochemical approaches, powerful information can be obtained about the structure and function of mitochondria in both animal model systems and human patients. While the described protocols refer to skeletal muscle and brain mitochondria, the methods described can be applied to any tissue type. © 2020 Elsevier Inc. All rights reserved.Assessment of the mitochondrial membrane potential (Δψ) in living cells, although not trivial, can be used to estimate mitochondrial bioenergetic state. For this purpose, fluorescent lipophilic cations are broadly applied. click here These cations enter cells and accumulate primarily in the mitochondrial matrix in a Δψ-dependent manner. Here, we describe the use of the cations tetramethylrhodamine methyl ester (TMRM) and rhodamine 123 (Rhod123) for semi-quantitative Δψ analysis in living mammalian cells. Two different strategies are presented (1) steady-state measurements that are suited to compare Δψ between different conditions (i.e., for comparing disease states or treatments) and (2) dynamic measurements allowing temporal monitoring of Δψ changes (i.e., to assess the effect of acute perturbations). We discuss the rationale for the use of TMRM and Rhod123 in their non-quenching/redistribution and quenching mode, how these modes are associated with different fluorescence responses, and how data can be interpreted. Practically, three experimental protocols are provided describing the use of TMRM and/or Rhod123 to assess Δψ in primary human skin fibroblasts (PHSFs) and neuron/astrocyte co-cultures by live-cell fluorescence microscopy. © 2020 Elsevier Inc. All rights reserved.Adenosine 5'-triphosphate (ATP) is the central metabolite in the energy metabolism of cells and is hydrolyzed to ADP and inorganic phosphate to provide free energy in various cellular processes. ATP also functions as an intracellular signaling molecule. Thus, it is important to know the ATP concentration within cells to understand cellular activities. Here, we describe two methods to detect ATP concentrations in the cytoplasm and mitochondrial matrix using genetically encoded luminescent or fluorescent biosensors. These methods enable quantitative investigation of ATP concentration dynamics in living cells, single cells and cell populations. © 2020 Elsevier Inc. All rights reserved.This review focuses on three independent and complementary approaches to obtain information on the combined function of respiratory complexes when present in different structural situations, either as individual complexes or when superassembled with other complexes. We review the utility of in-gel activity after blue native electrophoresis, integrated oxygen consumption of supercomplexes containing complex IV, and spectrophotometric activity measurements. © 2020 Elsevier Inc. All rights reserved.Mitochondria are responsible for the generation of ATP by oxidative phosphorylation; however, these multifaceted organelles regulate many other key cellular functions as well, such as calcium homeostasis, apoptosis, and biosynthesis of steroid hormones, heme and phospholipids. In order to carry out these functions, mitochondria establish physical and functional connections with other organelles such as the plasma membrane, lipid droplets/vesicles, peroxisomes, endosomes, and the endoplasmic reticulum. Dysregulation of any of the aforementioned processes or inter-organelle contacts can lead to mitochondrial dysfunction and subsequent changes in oxygen consumption and ATP production. Seahorse technology has become a critical tool for quantification of mitochondrial oxygen consumption and can help differentiate primary mitochondrial disorders from disorders where alterations in mitochondrial metabolism are consequences of a prior, upstream insult. In this chapter, we describe the application of Seahorse technology for assaying mitochondrial respiration in whole cells, permeabilized cells and isolated mitochondria. We leave it to the researcher's discretion to determine which of these approaches will generate the most physiologically relevant data based on the model system and research question at hand. © 2020 Elsevier Inc. All rights reserved.Measurement of the individual enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) forms a key part of diagnostic investigations in patients with suspected mitochondrial disease, and can provide crucial information on mitochondrial OXPHOS function in a variety of cells and tissues that are applicable to many research investigations. In this chapter, we present methods for analysis of mitochondrial respiratory chain enzymes in cells and tissues based on assays performed in two geographically separate diagnostic referral centers, as part of clinical diagnostic investigations. Techniques for sample preparation from cells and tissues, and spectrophotometric assays for measurement of the activities of OXPHOS complexes I-V, the combined activity of complexes II+III, and the mitochondrial matrix enzyme citrate synthase, are provided. The activities of mitochondrial respiratory chain enzymes are often expressed relative to citrate synthase activity, since these ratios may be more robust in accounting for variability that may arise due to tissue quality, handling and storage, cell growth conditions, or any mitochondrial proliferation that may be present in tissues from patients with mitochondrial disease.
Read More: https://www.selleckchem.com/products/Vorinostat-saha.html