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St. Jude Children's Research Hospital

13 ARTICLES PUBLISHED IN JoVE

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Medicine

Guidelines for Elective Pediatric Fiberoptic Intubation
Roland N. Kaddoum 1, Zulfiqar Ahmed 2, Alan A. D'Augsutine 2, Maria M. Zestos 3
1Department of Anesthesia, St. Jude Children's Research Hospital, 2Department of Anesthesia, Children's Hospital of Michigan, 3Department of Anesthesiology, Children's Hospital of Michigan

We describe guidelines to perform a safe and efficient elective fiberoptic intubation in pediatric patients while maintaining spontaneous ventilation.

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Biology

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)
Zhen Wang 1, Christoph Benning 1
1Department of Biochemistry and Molecular Biology, Michigan State University

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

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Neuroscience

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain
Ida Annunziata *1, Annette Patterson *1, Alessandra d'Azzo 1
1Department of Genetics, St Jude Children's Research Hospital

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

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Medicine

A Cell Culture Model of Resistance Arteries
Lauren A. Biwer 1,2, Christophe Lechauve 3, Sheri Vanhoose 4, Mitchell J. Weiss 3, Brant E. Isakson 1,2
1Department of Molecular Physiology and Biophysics, University of Virginia School of Medicine, 2Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 3Department of Hematology, St. Jude Children's Research Hospital, 4Research Histology Core, University of Virginia School of Medicine

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

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Biochemistry

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Anthony A. High 1, Haiyan Tan 1, Vishwajeeth R. Pagala 1, Mingming Niu 2,3, Ji-Hoon Cho 1, Xusheng Wang 1, Bing Bai 2, Junmin Peng 1,2
1St. Jude Proteomics Facility, St. Jude Children's Research Hospital, 2Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital, 3Heilongjiang University of Chinese Medicine

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

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Biology

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse
Zhen Wang 1,4, Ramon Ocadiz-Ruiz 1, Sinju Sundaresan 1, Lin Ding 1, Michael Hayes 1, Nirakar Sahoo 3, Haoxing Xu 1,2, Juanita L. Merchant 1,2,5
1Department of Internal Medicine-Gastroenterology, University of Michigan, 2Department of Molecular and Integrative Physiology, University of Michigan, 3Department of Molecular, Cellular and Developmental Biology, University of Michigan, 4Department of Gastrointestinal Surgery, The First Affiliated Hospital of Guangxi Medical University, 5Division of Gastroenterology, University of Arizona College of Medicine

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

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Biology

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis
Dan E. McNamara *1,2, Giovanni Quarato *3, Cliff S. Guy 3, Douglas R. Green 3, Tudor Moldoveanu 1,2
1Department of Structural Biology, St. Jude Children's Research Hospital, 2Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 3Department of Immunology, St. Jude Children's Research Hospital

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

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Biochemistry

Quantification of Coenzyme A in Cells and Tissues
Matthew W. Frank 1, Chitra Subramanian 1, Charles O. Rock 1, Suzanne Jackowski 1
1Department of Infectious Diseases, St. Jude Children's Research Hospital

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

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Biology

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts
Diantha van de Vlekkert 1, Xiaohui Qiu 1, Ida Annunziata 1, Alessandra d'Azzo 1
1Department of Genetics, St. Jude Children's Research Hospital

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

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Biology

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry
Zhen Wang *1, Kanisha Kavdia *2, Kaushik Kumar Dey 1, Vishwajeeth Reddy Pagala 2, Kiran Kodali 2, Danting Liu 1, Dong Geun Lee 1, Huan Sun 1, Surendhar Reddy Chepyala 1, Ji-Hoon Cho 2, Mingming Niu 1, Anthony A. High 2, Junmin Peng 1,2
1Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital, 2Center for Proteomics and Metabolomics, St. Jude Children's Research Hospital

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

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Biochemistry

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics
David Vanderwall 1, Poudel Suresh 1,2, Yingxue Fu 2, Ji-Hoon Cho 2, Timothy I. Shaw 2,3, Ashutosh Mishra 2, Anthony A. High 2, Junmin Peng 1,2, Yuxin Li 1,2
1Departments of Structural Biology and Developmental Neurobiology, St. Jude Children’s Research Hospital, 2Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, 3Department of Computational Biology, St. Jude Children’s Research Hospital

We present a systems biology tool JUMPn to perform and visualize network analysis for quantitative proteomics data, with a detailed protocol including data pre-processing, co-expression clustering, pathway enrichment, and protein-protein interaction network analysis.

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Medicine

Estimating Bilateral Atrial Function by Cardiovascular Magnetic Resonance Feature Tracking in Patients with Paroxysmal Atrial Fibrillation
Yanjing Wang 1, Hang Gao 1, Yi Li 1, Huan Sun *2, Lin Liu *1
1Radiology Department, China-Japan Union Hospital of Jilin University, 2Cardiology Department, China-Japan Union Hospital of Jilin University

The atrial function is associated with the strain and strain rate. The cardiac magnetic resonance feature tracking (CMR-FT) technique was used in this study to quantify left and right atrial global and segmental longitudinal strain and strain rate in individuals with paroxysmal atrial fibrillation.

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Immunology and Infection

Evaluation of Caspase Activation to Assess Innate Immune Cell Death
Joo-Hui Han 1, Rebecca E. Tweedell 1, Thirumala-Devi Kanneganti 1
1Department of Immunology, St. Jude Children's Research Hospital

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

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