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9 ARTICLES PUBLISHED IN JoVE

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Biology

DNA-based Fish Species Identification Protocol
Rachel Formosa 1, Harini Ravi 1, Scott Happe 1, Danielle Huffman 1, Natalia Novoradovskaya 1, Robert Kincaid 1, Steve Garrett 1
1Agilent Technologies

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

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Biology

Bioenergetic Profile Experiment using C2C12 Myoblast Cells
David G. Nicholls 1, Victor M. Darley-Usmar 2, Min Wu 3, Per Bo Jensen 3, George W. Rogers 3, David A. Ferrick 3
1Buck Institute for Age Research, Novato, CA, 2Department of Pathology, Center for Free Radical Biology, University of Alabama at Birmingham - UAB, 3Seahorse Bioscience, North Billerica, MA

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

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Medicine

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy
Manish S. Bharadwaj 1, Daniel J. Tyrrell 1, Mary F. Lyles 1, Jamehl L. Demons 1, George W. Rogers 2, Anthony J. A. Molina 1
1Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine, 2Seahorse Biosciences

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

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Developmental Biology

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements
Nabil E. Boutagy 1,2, George W. Rogers 3, Emily S. Pyne 1, Mostafa M. Ali 1, Matthew W. Hulver 1,2, Madlyn I. Frisard 1,2
1The Department of Human Nutrition, Foods, and Exercise, Virginia Tech, 2The Metabolic Phenotyping Core, Virginia Tech, 3Seahorse Bioscience

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

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Biology

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements
Nabil E. Boutagy 1,2, Emily Pyne 1, George W. Rogers 3, Mostafa Ali 1, Matthew W. Hulver 1,2, Madlyn I. Frisard 1,2
1The Department of Human Nutrition, Foods, and Exercise, Virginia Tech, 2The Metabolic Phenotyping Core, Virginia Tech, 3Seahorse Bioscience

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

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Cancer Research

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts
Pawan Noel 1, Ruben Muñoz 1, George W. Rogers 2, Andrew Neilson 2, Daniel D. Von Hoff 1, Haiyong Han 1
1Molecular Medicine Division, Translational Genomics Research Institute, 2Agilent Technologies

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

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Developmental Biology

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption
Joseph M. Elsakr 1, Charles Deeter 2, Valerie Ricciardi 3, Maureen Gannon 1,4,5,6
1Department of Molecular Physiology and Biophysics, Vanderbilt University, 2Agilent Technologies, 3Department of Biological Sciences, Vanderbilt University, 4Department of Veterans Affairs Tennessee Valley, 5Department of Medicine, Vanderbilt University Medical Center, 6Department of Cell and Developmental Biology, Vanderbilt University

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

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Biology

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C
Antonia Hauth 1, Rafael Galupa 1, Nicolas Servant 2, Laura Villacorta 1, Kai Hauschulz 3, Joke Gerarda van Bemmel 4, Agnese Loda 1, Edith Heard 1,5
1EMBL: European Molecular Biology Laboratory, 2Institut Curie, 3Agilent Technologies, 4Genmab BV, 5Collège de France

This protocol describes the Capture Hi-C method used to characterize the 3D organization of megabased-sized targeted genomic regions at high-resolution, including boundaries of topologically associating domains (TADs) and long-range chromatin interactions between regulatory and other DNA sequence elements.

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Cancer Research

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer
Kaitriana E. Colling *1,2, Emily L. Symons *1, Lorenzo Buroni *3, Hiruni K. Sumanasiri 1, Jessica Andrew-Udoh 1, Emily Witt 1,4, Haley A. Losh 1, Abigail M. Morrison 1, Kimberly K. Leslie 5, Christopher J. Dunnill 6, Johann S. de Bono 3, Kristina W. Thiel 1,7
1Department of Obstetrics and Gynecology, Carver College of Medicine, University of Iowa, 2Cancer Biology Graduate Program, Carver College of Medicine, University of Iowa, 3The Institute of Cancer Research: and the Royal Marsden NHS Foundation Trust, 4Department of Radiation Oncology, Carver College of Medicine, University of Iowa, 5Division of Molecular Medicine, Departments of Internal Medicine and Obstetrics and Gynecology, University of New Mexico Comprehensive Cancer Center, University of New Mexico Health Sciences Center, 6Agilent Technologies, 7Holden Comprehensive Cancer Center, University of Iowa

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

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