St. Jude Children's Research Hospital View Institution's Website 12 articles published in JoVE Immunology and Infection Evaluation of Caspase Activation to Assess Innate Immune Cell Death Joo-Hui Han1, Rebecca E. Tweedell1, Thirumala-Devi Kanneganti1 1 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. Biochemistry JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics David Vanderwall1, Poudel Suresh1,2, Yingxue Fu2, Ji-Hoon Cho2, Timothy I. Shaw2,3, Ashutosh Mishra2, Anthony A. High2, Junmin Peng1,2, Yuxin Li1,2 1Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, 3 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. Biology Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts Diantha van de Vlekkert1, Xiaohui Qiu1, Ida Annunziata1, Alessandra d'Azzo1 1 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. Biochemistry Quantification of Coenzyme A in Cells and Tissues Matthew W. Frank1, Chitra Subramanian1, Charles O. Rock1, Suzanne Jackowski1 1 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. Biology Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis Dan E. McNamara*1,2, Giovanni Quarato*3, Cliff S. Guy3, Douglas R. Green3, Tudor Moldoveanu1,2 1 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. Biochemistry Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification Anthony A. High1, Haiyan Tan1, Vishwajeeth R. Pagala1, Mingming Niu2,3, Ji-Hoon Cho1, Xusheng Wang1, Bing Bai2, Junmin Peng1,2 1 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. Medicine A Cell Culture Model of Resistance Arteries Lauren A. Biwer1,2, Christophe Lechauve3, Sheri Vanhoose4, Mitchell J. Weiss3, Brant E. Isakson1,2 1Department of Molecular Physiology and Biophysics, University of Virginia School of Medicine, 2Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 3 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. Medicine Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke Lora Talley Watts1, Wei Zheng1, R. Justin Garling2, Victoria C. Frohlich3, James Donald Lechleiter1 1Department of Cellular and Structural Biology, The University of Texas Health Science Center San Antonio, 2School of Medicine, The University of Texas Health Science Center San Antonio, 3 Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection. Neuroscience Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain Ida Annunziata*1, Annette Patterson*1, Alessandra d'Azzo1 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. Neuroscience Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction Rebecca Smith1, J. Paul Taylor1 1Developmental Neurobiology, St. Jude Children’s Research Hospital The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ. Medicine Guidelines for Elective Pediatric Fiberoptic Intubation Roland N. Kaddoum1, Zulfiqar Ahmed2, Alan A. D'Augsutine2, Maria M. Zestos3 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. Medicine A Novel Rescue Technique for Difficult Intubation and Difficult Ventilation Maria M. Zestos1, Dima Daaboul1, Zulfiqar Ahmed1, Nasser Durgham1, Roland Kaddoum2 1Department of Anesthesia, Children’s Hospital of Michigan, 2Department of Anesthesia, St. Jude Children’s Research Hospital We describe a technique to maintain oxygenation and ventilation using an endotracheal tube inserted nasally to the level of the naso-pharynx while sealing the mouth and nares for successful positive pressure ventilation.
