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6 ARTICLES PUBLISHED IN JoVE

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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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Immunology and Infection

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression
Zhihan Wang *1,2, Qinqin Pu *1,2, Ping Lin *2,3, Changlong Li 1, Jianxin Jiang 3, Min Wu 2
1West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, 2Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, 3State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Surgery Research, Daping Hospital, Army Medical University

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

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