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This volume presents a broad collection of state-of-the-art methods to study the biology of thermogenic fat using in vitro cell culture and animal models. Chapters guide the readers through protocols on differentiation of human pluripotent stem cells and murine adipocyte precursors; methods for measuring mitochondrial respiration, heat generation, brown fat activation, and effects on energy metabolism in mice; and techniques for AAV-mediated gene delivery, transplantation of adipose tissue, isolation of adipose tissue immune cells and extracellular vesicles, and mass spectrometry-based profiling of brown fat lipids. 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 methods, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge,Thermogenic Fat: Methods and Protocols aims to be comprehensive guide for researchers in the field.
List of contents
Differentiation of Human Pluripotent Stem cells (hPSCs) into Brown-Like Adipocytes.- Culture and Differentiation of Primary Preadipocytes from Mouse Subcutaneous White Adipose Tissue.- Isolation and Differentiation of Adipocyte Precursors Derived from Neonatal Murine Brown Adipose Tissue.- Determination of Adipocyte Size and Number.- Measuring Mitochondrial Uncoupling in Mouse Primary Brown Adipocytes Differentiated ex vivo.- Assessment of Bioenergetics in BAT Mitochondria.- Use of Isothermal Microcalorimetry to Measure Cellular Heat Production in Thermogenic Adipocytes.- Measurement of Intracellular Temperature in Brown Adipocytes using a Cationic Fluorescent Polymeric Thermometer.- Indirect Calorimetry to Assess Energy Balance in Mice: Measurement and Data Analysis.- Thermogenic Phenotyping in Mice.- Calculating Diet-Induced Thermogenesis in Mice.- Assessment of Brown and Beige Adipose Tissue Activation in Mice using PET/CT Imaging.- Imaging Brown Adipose Tissue with TSPO PET Tracers in Preclinical Animal Studies.- Use of CIDEA Reporter Mouse Model for Screening Thermogenic Fat Activating Drugs.- AAV-Mediated Gene Delivery to Mouse Brown Adipose Tissue.- Subcutaneous Transplantation of White Adipose Tissue.- Brown Adipose Tissue Transplantation.- Isolation and Characterization of Brown Adipose Tissue T Cells.- Isolation of Adipose Tissue Extracellular Vesicles.- Isolation and Mass Spectrometry-Based Profiling of Major Lipids in Brown Adipose Tissue.- Methods for Single Cell Transcriptomic Analysis of Adipose Tissue.
