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丁香实验推荐阅读
Molecular Profiling of Mitochondrial Dysfunction in Caenorhabditis elegans

Cellular effects of primary mitochondrial dysfunction, as well as potential mitochondrial disease therapies, can be modeled in living animals such as the microscopic nematode, Caenorhabditis elegans. In particular, molecular analyses can provide substantial insight into t ...

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Fluorescence-Activated Cell Sorting Analysis of Mitochondrial Content, Membrane Potential, and Matrix Oxidant Burden in Human Lymphoblastoid Cell Line

Fluorescence-activated cell sorting (FACS) permits specific biologic parameters of cellular populations to be quantified in a high-throughput fashion based on their unique fluorescent properties. Relative quantitation of mitochondrial-localized dyes in human cells u ...

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Measurement of Oxidized and Reduced Coenzyme Q in Biological Fluids, Cells, and Tissues: An HPLC-EC Method

Direct measure of coenzyme Q (CoQ) in biological specimens may provide important advantages. Precise and selective high-performance liquid chromatography (HPLC) methods with electrochemical (EC) detection have been developed for the measurement of reduced (ubiquinol) and o ...

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Assay to Measure Oxidized and Reduced Forms of CoQ by LCMS/MS

The redox status of mitochondrial coenzyme Q (CoQ) is an important marker for oxidative stress associated with several disorders such as Parkinson disease and Alzheimer disease. Altered redox status may be present in mitochondrial electron transport complex disorders. Intracel ...

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Blue Native Polyacrylamide Gel Electrophoresis: A Powerful Diagnostic Tool for the Detection of Assembly Defects in the Enzyme Complexes of Oxidative

The bulk of ATP consumed by various cellular processes is normally produced by five multimeric protein complexes embedded within the inner mitochondrial membrane in a process known as oxidative phosphorylation (OXPHOS). Mutations that impair the assembly, and therefore the funct ...

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Morphological Assessment of Mitochondrial Respiratory Chain Function on Tissue Sections

In recent decades, genetic, biochemical, immunological, and cell biological techniques have been applied not only for better understanding of pathogenesis of known mitochondrial encephalomyopathies but also for exploring the possibility of mitochondrial involvement in o ...

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Radioactive Labeling of Mitochondrial Translation Products in Cultured Cells

The mammalian mitochondrial genome contains 37 genes, 13 of which encode polypeptide subunits in the enzyme complexes of the oxidative phosphorylation system. The other genes encode the rRNAs and tRNAs necessary for their translation. The mitochondrial translation machinery is l ...

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Transmitochondrial Cybrids: Tools for Functional Studies of Mutant Mitochondria

Mitochondrial functions are controlled by both mitochondrial DNA (mtDNA) and nuclear DNA. Hence, it is difficult to identify whether mitochondrial or nuclear genome is responsible for a particular mitochondrial defect. Cybrid is a useful tool to overcome this difficulty, where we can c ...

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Quantification of mtDNA Mutation Heteroplasmy (ARMS qPCR)

Pathogenic mitochondrial DNA (mtDNA) mutations are usually present in heteroplasmic forms that vary in concentration among different tissues. Manifestation of clinical phenotypes depends on the degree of mtDNA mutation heteroplasmy (mutation load) in affected tissues. It is t ...

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Determination of the Clinical Significance of an Unclassified Variant

After completion of Human Genome Project (HGP) in 2003, as well as the new technology development in genomic research, the most accurate genetics blueprint of human is available. Researchers started to dissect and understand the genetic map of the human species. As a consequence, analyses of no ...

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Measurement of Mitochondrial DNA Copy Number

Mitochondrial disorders are complex and heterogeneous diseases that may be caused by molecular defects in either the nuclear or mitochondrial genome. The biosynthesis and maintenance of the integrity of the mitochondrial genome is solely dependent on a number of nuclear proteins. De ...

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Optimized Protocols to Analyze Sphingosine-1-Phosphate Signal Transduction Pathways During Acrosomal Exocytosis in Human Sperm

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. Sphingosine 1-phosphate is a bioactive sphingolipid that regulates crucial physiological processes. We have r ...

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Quantifying Sphingosine-1-Phosphate-Dependent Activation of the RhoGTPases

The GPCR-coupled sphingosine-1-phosphate (S1P) receptors regulate a number of important cell �functions, including proliferation, migration, and adhesion. Since these processes require dynamic regulation of the actin cytoskeleton, the ability to monitor S1P-dependent a ...

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High-Throughput Assays to Measure Intracellular Ca2+ Mobilization in Cells that Express Recombinant S1P Receptor Subtypes

Intracellular Ca2+ mobilization is a useful readout to screen for agonists or antagonists of G-protein �coupled receptors (GPCRs). Here, we describe methods to conduct high-throughput screening of stably or transiently transfected HTC4 cells expressing the individual S1P1–5 re ...

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Assessment of Sphingosine-1-Phosphate Activity in Biological Samples by Receptor Internalization and Adherens Junction Formation

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator involved in many biological actions, including vascular homeostasis and immune cell trafficking. S1P activity is mediated by specific G protein-coupled receptors, leading to multiple physiological responses in ...

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Immunohistochemical Detection of Sphingosine-1-Phosphate and Sphingosine Kinase-1 in Human Tissue Samples

Sphingosine-1-phosphate (S1P) and the enzyme primarily responsible for its production, sphingosine kinase-1 (SphK-1), are thought to be dysregulated in multiple human diseases including cancer, multiple sclerosis (MS), diabetes, neurological diseases, fibrosis, and certa ...

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A Cleanup Method for Mass Spectrometry of Sphingosine-1-Phosphate in Blood and Solid Tissues Using a Phosphate Capture Molecule

Cleanup technology and mass spectrometric determination of sphingosine-1-phosphate using a �phosphate capture molecule are shown. The protocol is rapid, requires neither thin-layer chromatography nor liquid chromatography, and is applicable to both blood and solid tissue s ...

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Quantification of Sphingosine-1-Phosphate and Related Sphingolipids by Liquid Chromatography Coupled to Tandem Mass Spectrometry

Liquid chromatography coupled to tandem mass spectrometry has evolved as the method of choice for the detection of sphingolipid metabolites due to its high sensitivity and superior specificity compared to other methodological approaches. Here, we describe a simple and robust method ...

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Isoform-Selective Assays for Sphingosine Kinase Activity

Sphingosine kinases (SK) 1 and 2 are unique lipid kinases that phosphorylate sphingosine to form �sphingosine-1-phosphate (S1P). S1P is a bioactive molecule eliciting multiple effects both extracellularly via cell surface S1P receptors and intracellularly through a number of re ...

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Biochemical Methods for Quantifying Sphingolipids: Ceramide, Sphingosine, Sphingosine Kinase-1 Activity, and Sphingosine-1-Phosphate

Sphingolipids (ceramide, sphingosine, and sphingosine-1-phosphate) are bioactive lipids with important biological functions in proliferation, apoptosis, angiogenesis, and inflammation. Herein, we describe easy and rapid biochemical methods with the use of radiolabe ...

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