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This volume details comprehensive protocols and methodologies to assess mitochondrial bioenergetics and dynamics in different tissues and cells involving health and pathological states. Chapters guide readers through methods for assessment of the energy metabolism including Oxygen Consumption Rate (OCR), mitochondrial membrane potential, and measuring mitochondrial Ca2+ handling, and ROS emission. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting, and systematic reproducible protocols.
Authoritative and cutting-edge, Mitochondria: Methods and Protocols aims to be a foundation for future studies and to be a source of inspiration for new investigations in the field.
List of contents
Fast Determination of Mitochondrial Metabolism and Respiratory Complex Activity in Permeabilized and Intact Cells.- Unbiased Millivolts Assay of Mitochondrial Membrane Potential in Intact Cells.- Rotenone Decreases Ischemia-induced Injury by Inhibiting Mitochondrial Permeability Transition - A study in Brain.- Assessment of Mitochondrial Complex II and III Activity In Brain Sections: a Histoenzymological Technique.- Measurement of mitochondrial (dys)function in cellular systems using Electron Paramagnetic Resonance (EPR): oxygen consumption rate and superoxide production.- Mitochondrial Calcium Handling in Isolated Mitochondria from a Guinea Pig Heart.- Characterizing the Electron Transport Chain---Structural Approach.- Characterizing the electron transport chain--functional approach using Extracellular Flux Analyzer on mouse tissue samples.- Simultaneous Acquisition of Mitochondrial Calcium Retention Capacity and Swelling to Measure Permeability Transition Sensitivity.- Measuring mitochondrial Function: From organelle to organism.- Mitochondrial Toxicity of Organic Arsenicals.- Measurements of Mitochondrial Respiration in Intact Cells, Permeabilized Cells, And Isolated Tissue Mitochondria using the Seahorse XF Analyzer.- Monitoring mitochondrial morphology and respiration in Doxorubicin-induced cardiomyopathy.- Creation of yeast models for evaluating the pathogenicity of mutations in the human mitochondrial gene MT-ATP6 and discovering therapeutic molecules.- In vivo analysis of mitochondrial protein synthesis in Saccharomyces cerevisiae mitochondrial tRNA mutants.- Visualizing mitochondrial importability of a protein using the yeast Bi-genomic Mitochondrial-Split-GFP strain and an ordinary fluorescence microscope.- Analysis of Mitochondrial Performance in Lymphocytes using Fluorescent Life--Time Imaging Microscopy.- Monitoring mitochondrial perturbationsduring infection.- Measuring the Mitochondrial Ubiquinone (Q) Pool Redox State In Isolated Respiring Mitochondria.- Experimental Setup For Investigation Of Acute Mitochondrial Oxygen Sensing In Primary Cells.- Assessing the redox status of mitochondria through the NADH/FAD+ ratio in intact cells.- Monitoring mitochondrial membrane potential in live cells using time-lapse fluorescence imaging.- Investigating mitochondrial Ca2+ dynamics in isolated mitochondria and intact cells: Application of fluorescent dyes and genetic reporters.- A plate reader-based measurement of the cellular ROS production using Dihydroethidium and MitoSOX.- Analysis of organisation and activity of mitochondrial respiratory chain complexes in primary fibroblasts using blue native PAGE.- Multiplexing Seahorse XFe24 and ImageXpress® Nano platforms for comprehensive evaluation of mitochondrial bioenergetic profile and neuronal morphology.- Computational Modelling of Mitochondria to Understand the Dynamics of Oxidative Stress.
