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University of Pittsburgh

91 ARTICLES PUBLISHED IN JoVE

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Biology

Development of automated imaging and analysis for zebrafish chemical screens.
Andreas Vogt 1, Hiba Codore 2, Billy W. Day 3,4, Neil A. Hukriede 2, Michael Tsang 2
1Pharmacology and Chemical Biology, University of Pittsburgh Drug Discovery Institute, 2Department of Microbiology and Molecular Genetics, University of Pittsburgh, 3Department of Pharmaceutical Sciences, University of Pittsburgh, 4Department of Chemistry, University of Pittsburgh

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

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Biology

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos
Jian Zou 1, Xiangyun Wei 1,2
1Department of Ophthalmology, University of Pittsburgh, 2Department of Microbiology and Molecular Genetics, University of Pittsburgh

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

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Biology

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis
Sagar D. Joshi 1, Lance A. Davidson 1,2
1Bioengineering, University of Pittsburgh, 2Developmental Biology, University of Pittsburgh

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

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Biology

Isolating Stem Cells from Soft Musculoskeletal Tissues
Yong Li 1,2,3,4, Haiying Pan 1, Johnny Huard 1,2,3,4,5
1Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC, 2Department of Bioengineering, University of Pittsburgh, 3Department of Orthopedic Surgery, University of Pittsburgh, 4Department of Pathology, University of Pittsburgh, 5Department of Molecular Genetics & Biochemistry, University of Pittsburgh

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

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Medicine

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock
Lauryn K. Kohut 1, Sophie S. Darwiche 1, John M. Brumfield 1, Alicia M. Frank 1, Timothy R. Billiar 1
1Department of Surgery, University of Pittsburgh

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

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Medicine

Pseudofracture: An Acute Peripheral Tissue Trauma Model
Sophie S. Darwiche 1, Philipp Kobbe 2, Roman Pfeifer 2, Lauryn Kohut 1, Hans-Christoph Pape 2, Timothy Billiar 1
1Department of Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Aachen Medical Center

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

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Biology

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury
Chiara Cianciolo Cosentino 1, Beth L. Roman 2, Iain A. Drummond 3, Neil A. Hukriede 1
1Department of Developmental Biology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh, 3Department of Medicine and Genetics, Harvard Medical School

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

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Biology

Generation of Transgenic C. elegans by Biolistic Transformation
Daniel Hochbaum 1, Annabel A. Ferguson 1, Alfred L. Fisher 1
1Department of Medicine, University of Pittsburgh

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

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Biology

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker
Yue Zhang *1, Luv Kashyap *2, Annabel A. Ferguson 2, Alfred L. Fisher 2
1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

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Medicine

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface
Hilaire C. Lam 1,2, Augustine M.K. Choi 1, Stefan W. Ryter 1
1Department of Medicine, Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, 2Cellular and Molecular Pathology, School of Medicine, University of Pittsburgh

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

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Bioengineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering
Kee-Won Lee 1, Yadong Wang 1,2
1Department of Bioengineering, University of Pittsburgh, 2McGowan Institute for Regenerative Medicine, University of Pittsburgh

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

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Immunology and Infection

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Helene Mens 1, Mary Kearney 1, Ann Wiegand 1, Jonathan Spindler 1, Frank Maldarelli 1, John W. Mellors 2, John M. Coffin 3
1The virology Core at the HIV Drug Resistance Program, NCI-Frederick, 2Division of Infectious Diseases, University of Pittsburgh, 3Department of Molecular Biology and Microbiology, Tuffts University

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

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Biology

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach
Maria Jaramillo 1, Ipsita Banerjee 1,2
1Department of Bioengineering, University of Pittsburgh, 2Department of Chemical Engineering, University of Pittsburgh

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

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Immunology and Infection

Growth of Mycobacterium tuberculosis Biofilms
Kathleen Kulka 1, Graham Hatfull 2, Anil K. Ojha 1
1Department of Infectious Diseases and Microbiology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

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Medicine

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation
Fabrisia Ambrosio 1,2,3, G. Kelley Fitzgerald 2, Ricardo Ferrari 1,2, Giovanna Distefano 1,2, George Carvell 2
1Department of Physical Medicine and Rehabilitation, University of Pittsburgh, 2Department of Physical Therapy, University of Pittsburgh, 3McGowan Institute for Regenerative Medicine, University of Pittsburgh

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

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Neuroscience

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging
Jeffrey S. Phillips 1,2, Adam S. Greenberg 1,3, John A. Pyles 1,3, Sudhir K. Pathak 1,4, Marlene Behrmann 1,3, Walter Schneider 1,3, Michael J. Tarr 1,3
1Center for the Neural Basis of Cognition, 2Department of Psychology, University of Pittsburgh, 3Department of Psychology, Carnegie Mellon University , 4Department of Bioengineering, University of Pittsburgh

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

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Clinical Skills

Accessibility to Clinical Care for People who use Wheelchairs
Yetsa Tuakli-Wosornu 1,2, Jaideep Talwalkar 1
1Yale School of Medicine, 2University of Pittsburgh

Accessibility to Clinical Care for People who use Wheelchairs

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Clinical Skills

Physical Exam Considerations for People who use Wheelchairs
Yetsa Tuakli-Wosornu 1,2, Jaideep Talwalkar 1
1Yale School of Medicine, 2University of Pittsburgh

Physical Exam Considerations for People who use Wheelchairs

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Bioengineering

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion
Nathaniel T. Remlinger 1,2, Peter D. Wearden 1,3, Thomas W. Gilbert 1,2,3,4
1McGowan Institute for Regenerative Medicine, 2Department of Bioengineering, University of Pittsburgh, 3Department of Cardiothoracic Surgery, Children's Hospital of Pittsburgh of UPMC, 4Department of Surgery, University of Pittsburgh

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

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Biology

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia
Richard Francis 1, Cecilia Lo 1
1Department of Developmental Biology, University of Pittsburgh

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

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Immunology and Infection

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP
Hongshuai Li 1,2, Bingyun Li 1,3,4
1Department of Orthopaedics, School of Medicine, West Virginia University , 2Department of Orthopaedics, Stem Cell Research Center, University of Pittsburgh, 3WVNano Initiative, 4Mary Babb Randolph Cancer Center

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

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Engineering

Synthesis and Functionalization of Nitrogen-doped Carbon Nanotube Cups with Gold Nanoparticles as Cork Stoppers
Yong Zhao 1, Yifan Tang 1, Alexander Star 1
1Department of Chemistry, University of Pittsburgh

We discussed the synthesis of individual graphitic nanocups using a series of techniques including chemical vapor deposition, acid oxidation and probe-tip sonication. By citrate reduction of HAuCl4, the graphitic nanocups were effectively corked with gold nanoparticles due to the chemically reactive edges of the cups.

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JoVE Core

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes
Laura S. Kocsis 1, Erica Benedetti 1, Kay M. Brummond 1
1Department of Chemistry, University of Pittsburgh

Microwave-assisted intramolecular dehydrogenative Diels-Alder (DA) reactions provide concise access to functionalized cyclopenta[b]naphthalene building blocks. The utility of this methodology is demonstrated by one-step conversion of the dehydrogenative DA cycloadducts into novel solvatochromic fluorescent dyes via Buchwald-Hartwig palladium-catalyzed cross-coupling reactions.

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Immunology and Infection

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice
Ilias I. Siempos 1,3, Hilaire C. Lam 1, Yan Ding 2, Mary E. Choi 2, Augustine M. K. Choi 1, Stefan W. Ryter 1
1Department of Medicine, Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, 2Department of Medicine, Renal Division, Brigham and Women's Hospital, Harvard Medical School, 3First Department of Critical Care Medicine and Pulmonary Services, University of Athens Medical School, Evangelismos Hospital, Athens, Greece

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

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Immunology and Infection

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians
Laura A. Brannelly 1
1School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University

Batrachochytrium dendrobatidis infection in amphibians can be eliminated using a reduced concentration (0.0025%) and shorter duration (five minute baths for six days) of itraconazole than is generally used. Less mortality and fewer negative side effects were observed using this modified treatment protocol.

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Biology

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle
William C.W. Chen 1, Arman Saparov 2,3, Mirko Corselli 4, Mihaela Crisan 5, Bo Zheng 6, Bruno Péault 7,8, Johnny Huard 9
1Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Pittsburgh, 3Nazarbayev University Research and Innovation System, Nazarbayev University, 4Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 5Department of Cell Biology, Erasmus MC Stem Cell Institute, 6OHSU Center for Regenerative Medicine, Oregon Health & Science University, 7Centre for Cardiovascular Science and MRC Centre for Regenerative Medicine, Queen's Medical Research Institute and University of Edinburgh, 8David Geffen School of Medicine and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 9Stem Cell Research Center, Department of Orthopedic Surgery and McGowan Institute for Regenerative Medicine, University of Pittsburgh

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

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Medicine

A Standardized Obstacle Course for Assessment of Visual Function in Ultra Low Vision and Artificial Vision
Amy Catherine Nau 1, Christine Pintar 1, Christopher Fisher 1, Jong-Hyeon Jeong 2, KwonHo Jeong 2
1UPMC Eye Center, University of Pittsburgh, 2Department of Biostatistics, University of Pittsburgh

We describe an indoor, portable, standardized course that can be used to evaluate obstacle avoidance in persons who have ultralow vision. The course is relatively inexpensive, simple to administer, and has been shown to be reliable and reproducible.

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Engineering

Writing and Low-Temperature Characterization of Oxide Nanostructures
Akash Levy 1, Feng Bi 1, Mengchen Huang 1, Shicheng Lu 1, Michelle Tomczyk 1, Guanglei Cheng 1, Patrick Irvin 1, Jeremy Levy 1
1Department of Physics, University of Pittsburgh

Oxide nanostructures provide new opportunities for science and technology. The interfacial conductivity between LaAlO3 and SrTiO3 can be controlled with near-atomic precision using a conductive atomic force microscopy technique. The protocol for creating and measuring conductive nanostructures at LaAlO3/SrTiO3 interfaces is demonstrated.

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Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings
Bin Feng 1, G.F. Gebhart 1
1Center for Pain Research, Department of Anesthesiology, University of Pittsburgh

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

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Medicine

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye
Larry Kagemann 1,2, Bo Wang 2, Gadi Wollstein 1, Hiroshi Ishikawa 1,2, Brandon Mentley 1, Ian Sigal 1,2,3, Richard A Bilonick 1,4, Joel S Schuman 1,2,3
1Department of Ophthalmology, UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, 2Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, 3The McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, 4Deptartment of Biostatistics, Graduate School of Public Health, University of Pittsburgh

Trabecular meshwork (TM) migration into Schlemm’s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

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Immunology and Infection

Isolation and Transplantation of Different Aged Murine Thymic Grafts.
Y. Maurice Morillon II *1, Fatima Manzoor *1, Bo Wang 1, Roland Tisch 1
1Department of Microbiology and Immunology, University of North Carolina at Chapel Hill

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

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JoVE Journal

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration
Tae-Young Choi 1, Mehwish Khaliq 1, Sungjin Ko 1, Juhoon So 1, Donghun Shin 1
1Department of Developmental Biology, McGowan Institute for Regenerative Medicine, University of Pittsburgh

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

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Medicine

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms
Manav Korpal 1, Jacob Feala 1, Xiaoling Puyang 1, Jian Zou 1, Alex H. Ramos 2, Jeremy Wu 1, Timm Baumeister 1, Lihua Yu 1, Markus Warmuth 1, Ping Zhu 1
1H3 Biomedicine, 2Massachusetts General Hospital

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

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Developmental Biology

Quantification of Colonic Stem Cell Mutations
Ryan D. Whetstone 1, Barry Gold 1
1Department of Pharmaceutical Sciences, University of Pittsburgh

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

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Biology

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1
Karthika Natarajan 1,2, Yi Xie 1,3, Takeo Nakanishi 4, Rebecca S. Moreci 5,6, Pancharatnam Jeyasuria 7, Arif Hussain 1,3,8,9, Douglas D. Ross 1,3,8,9,10,11
1Greenebaum Cancer Center, University of Maryland School of Medicine, 2Pharmaceutical Sciences, University of Maryland School of Pharmacy, 3Baltimore VA Medical Center, 4Membrane Transport and Biopharmaceutics, School of Pharmaceutical Sciences, Kanazawa University, 5Obstetrics, Gynecology and Reproductive Science, University of Pittsburgh, 6Magee Women's Research Institute, 7Obstetrics, Gynecology, Perinatal Research Branch (NICHD), Wayne State University School of Medicine, 8Medicine, University of Maryland School of Medicine, 9Pathology, University of Maryland School of Medicine, 10Pharmacology, University of Maryland School of Medicine, 11Experimental Therapeutics, University of Maryland School of Medicine

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

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Medicine

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side
Stefanie Dinner 1, Julia Borkowski 1, Carolin Stump-Guthier 1, Hiroshi Ishikawa 2, Tobias Tenenbaum 1, Horst Schroten 1, Christian Schwerk 1
1Department of Pediatrics, Medical Faculty Mannheim, Heidelberg University, 2Department of NDU Life Sciences, Nippon Dental University

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

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Biology

FRET Imaging in Three-dimensional Hydrogels
Amalie E. Donius *1, Sylvain V. Bougoin *1,3, Juan M. Taboas 1,2
1Department of Oral Biology, Center for Craniofacial Regeneration, McGowan Institute of Regenerative Medicine, University of Pittsburgh, 2Department of Bioengineering, Center for Craniofacial Regeneration, McGowan Institute of Regenerative Medicine, University of Pittsburgh, 3Laerdal AS

Förster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

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Developmental Biology

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue
James E. Baily 1, William C.W. Chen 2,3, Nusrat Khan 4, Iain R. Murray 4, Zaniah N. González Galofre 4, Johnny Huard 5,6, Bruno Péault 4,7
1Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 2Department of Bioengineering and Orthopaedic Surgery, University of Pittsburgh, 3Research Laboratory of Electronics and Department of Biological Engineering, Massachusetts Institute of Technology, 4MRC Centre for Regenerative Medicine, University of Edinburgh, 5Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, 6Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, 7Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, David Geffen School of Medicine, University of California at Los Angeles

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

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Biology

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations
Huazheng Liang *1,2, Bassem Akladios *1, Cesar P. Canales *1, Richard Francis 3, Edna H. Hardeman 1, Annemiek Beverdam 1
1The School of Medical Sciences, University of New South Wales Australia, 2Neuroscience Research Australia, 3Biomedical Imaging Facility, University of New South Wales Australia

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

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Biochemistry

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins
Jenna K. Zalewski 1, Simone Heber 1,2, Joshua H. Mo 1, Keith O'Conor 1, Jeffrey D. Hildebrand 1, Andrew P. VanDemark 1
1Department of Biological Sciences, University of Pittsburgh, 2Institute of Structural Biology, German Research Center for Environmental Health

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

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Bioengineering

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials
Keith C. Heyde *1,2, Felicia Y. Scott *3, Sung-Ho Paek 3, Ruihua Zhang 3, Warren C. Ruder 3,4
1Department of Mechanical Engineering, Carnegie Mellon University, 2Engineering Science and Mechanics Program, Virginia Polytechnic Institute and State University, 3Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 4Department of Bioengineering, University of Pittsburgh

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

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Biochemistry

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)
Christina D. King 1, Joseph D. Dudenhoeffer 1, Liqing Gu 2, Adam R. Evans 3, Renã A. S. Robinson 1
1Department of Chemistry, University of Pittsburgh, 2SGS North America Inc., 3Large Molecule Analytical Development, Pharmaceutical Development & Manufacturing Science, Janssen Research and Development

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

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Medicine

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring
Mitchell Dyer 1, Shannon Haldeman 1, Andres Gutierrez 1, Lauryn Kohut 1, Anirban Sen Gupta 2, Matthew D. Neal 1
1Department of Surgery, University of Pittsburgh, 2Department of Biomedical Engineering, Case Western Reserve University

Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model.

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Neuroscience

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0
Rashed Harun 1,2,3, Christine M. Grassi 2, Miranda J. Munoz 2,4, Amy K. Wagner 1,2,3
1Center for Neuroscience, University of Pittsburgh, 2Department of Physical Medicine & Rehabilitation, University of Pittsburgh, School of Medicine, 3Safar Center for Resuscitation Research, University of Pittsburgh, 4Department of Biological Sciences, Mellon College of Science, Carnegie Mellon University

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

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Cancer Research

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization
Frank J. Jenkins 1,2, Charles M. Kerr 2, Elise Fouquerel 2,3, Dana H. Bovbjerg 2,4, Patricia L. Opresko 2,3
1Departments of Pathology and Infectious Diseases and Microbiology, University of Pittsburgh, 2University of Pittsburgh Cancer Institute, 3Department of Environmental and Occupational Health, University of Pittsburgh, 4Departments of Psychiatry, Psychology, Behavioral & Community Health Sciences, University of Pittsburgh

In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length.

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Biology

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis
Shinichiro Yokota 1,2, Yoshihiro Ono 2, Toshimasa Nakao 2, Peng Zhang 3, George K. Michalopoulos 4,5, Zahida Khan 3,4,5,6
1Department of Surgery, Jichi Medical University, 2Department of Surgery, University of Pittsburgh School of Medicine, 3Department of Pediatrics, University of Pittsburgh School of Medicine, 4Department of Pathology, University of Pittsburgh School of Medicine, 5Pittsburgh Liver Research Center, University of Pittsburgh, 6McGowan Institute for Regenerative Medicine, University of Pittsburgh

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

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Environment

Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity
Jack E. Watson 1, Tyson Robb 2, Danielle Andrews-Brown 3, Melissa Miller 1
1Department of Ecosystem Science and Management, Pennsylvania State University, 2Department of Geography and Environmental Sustainability, State University of New York, Oneonta, 3Department of Geology and Environmental Science, University of Pittsburgh

Here we present a methodology which matches a soil sample size and a hydraulic conductivity measurement device to prevent the so-called wall flow along the inside of the soil container from being erroneously included in water flow measurements. Its use is demonstrated with samples collected from a wastewater irrigation site.

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Immunology and Infection

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis
Laura A. Brannelly 1,2, Alexandra A. Roberts 1, Lee F. Skerratt 1, Lee Berger 1
1One Health Research Group, College of Public Health, Medical and Veterinary Sciences, James Cook University, 2Department of Biological Sciences, University of Pittsburgh

We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

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Medicine

Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion
Despoina Aslanoglou 1, Emily W. George 1, Zachary Freyberg 1,2
1Department of Psychiatry, University of Pittsburgh, 2Department of Cell Biology, University of Pittsburgh

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

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Bioengineering

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications
Ming-Cheng Chen *1, John R. Lake *1, Keith C. Heyde 2, Warren C. Ruder 1,2
1Department of Bioengineering, University of Pittsburgh, 2Department of Mechanical Engineering, Carnegie Mellon University

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

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Immunology and Infection

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics
Hyunryul Ryu 1, Kyungyong Choi 1,2, Yanyan Qu 3, Taehong Kwon 1,2, Janet S. Lee 3,5, Jongyoon Han 1,2,4
1Research Laboratory of Electronics, Massachusetts Institute of Technology, 2Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 3Department of Medicine, University of Pittsburgh, 4Department of Biological Engineering, Massachusetts Institute of Technology, 5Vascular Medicine Institute, University of Pittsburgh

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

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JoVE Core

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing
Justin N. Williams 1, Yong Li 1, Anuradha Valiya Kambrath 1, Uma Sankar 1
1Department of Anatomy and Cell Biology, Indiana University School of Medicine

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

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Medicine

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney
Brittney M. Rush *1, Sarah A Small *1, Donna B. Stolz 2, Roderick J. Tan 1
1Division of Renal-Electrolyte, Department of Medicine, University of Pittsburgh, 2Department of Cell Biology, University of Pittsburgh

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

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Biology

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face
Anne M. Burrows 1,2, Kailey M Omstead 1, Ashley R Deutsch 3,5, Justin T. Gladman 4, Adam Hartstone-Rose 3
1Department of Physical Therapy, Duquesne University, 2Department of Anthropology, University of Pittsburgh, 3College of Sciences, North Carolina State University, 4Shared Materials Instrumentation Facility, Duke University, 5Department of Anthropology, University of Florida

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

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Chemistry

A Rhodopsin Transport Assay by High-Content Imaging Analysis
Bing Feng 1, Xujie Liu 1, Yuanyuan Chen 1,2
1Department of Ophthalmology, University of Pittsburgh, 2McGowan Institute for Regenerative Medicine, University of Pittsburgh

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

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Medicine

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice
Sidney Mahan 1, Mingjun Liu 1, Richard A. Baylis 2,3, Delphine Gomez 1,4
1Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, 2Robert M. Berne Cardiovascular Research Center, University of Virginia, 3Department of Biochemistry and Molecular Genetics, University of Virginia, 4Division of Cardiology, University of Pittsburgh School of Medicine

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

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Neuroscience

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice
Liang Wen *1, Wendong You *1, Yadong Wang *1, Yuanrun Zhu 1, Hao Wang 1, Xiaofeng Yang 1
1Emergency and Trauma Center, The International Medical Center, The First Affiliated Hospital, School of Medicine, Zhejiang University

Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.

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Cancer Research

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury
Hamza O. Yazdani 1, Samer Tohme 1
1Department of Surgery, University of Pittsburgh

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

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Education

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency
Marianne Theile 1, Linus Wiora 1, Dominik Russ 1, Jonas Reuter 1, Hiroshi Ishikawa 2, Christian Schwerk 3, Horst Schroten 3, Stefan Mogk 1
1Interfaculty Institute of Biochemistry, University of Tübingen, 2Laboratory of Clinical Regenerative Medicine, Department of Neurosurgery, Faculty of Medicine, University of Tsukuba, 3Department of Pediatrics, Medical Faculty Mannheim, Heidelberg University

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

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Medicine

Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction
Anna L. Lang 1,2, William T. Goldsmith 3,4, Regina D. Schnegelberger 5,6, Gavin E. Arteel 6,7, Juliane I. Beier 6,7
1Department of Pharmacology and Toxicology, University of Louisville, 2Hepatobiology and Toxicology Program, University of Louisville, 3Department of Physiology and Pharmacology, West Virginia University, 4Center for Inhalation Toxicology, West Virginia University, 5Department of Pharmacology and Chemical Biology, University of Pittsburgh, 6Pittsburgh Liver Research Center, University of Pittsburgh, 7Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh

The goal of this protocol was to develop a murine model of low-level toxicant exposure that does not cause overt liver injury but rather exacerbates pre-existing liver damage. This paradigm better recapitulates human exposure and the subtle changes that occur upon exposure to toxicant concentrations that are considered safe.

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Biology

Measuring RAN Peptide Toxicity in C. elegans
Paige Rudich 1, Carley Snoznik 2, Noah Puleo 2, Todd Lamitina 1,2
1Graduate Program in Cell Biology and Molecular Physiology, University of Pittsburgh, 2Department of Pediatrics, University of Pittsburgh School of Medicine

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

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Bioengineering

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy
Ying Yin *1, Siyu Wang *1, Danni Hu 1, Jingyao Cai 1, Fubin Chen 1, Bo Wang 1, Yu Gao 1
1Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

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Immunology and Infection

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes
Wei Hu 1,2, Xiang Song 1, Haibo Yu 1, Jingyu Sun 2, Yong Zhao 1
1Center for Discovery and Innovation, Hackensack Meridian Health Center, 2Department of Chemistry and Chemistry Biology, Stevens Institute of Technology

Exosome application is an emerging tool for drug development and regenerative medicine. We establish an exosome isolation protocol with high purity to isolate exosomes from novel identified stem cells called CB-SC for mechanistic studies. We also coculture CB-SC-derived exosomes with human monocytes, leading to their differentiation into phenotypically distinct macrophages.

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Immunology and Infection

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients
Lindsay Jackson 1, William DePas 2, Amanda J. Morris 1, Kevin Guttman 1, Yvonne C W Yau 1,3, Valerie Waters 1,4
1Translational Medicine, Hospital for Sick Children, 2Center for Microbial Pathogenesis, Department of Pediatrics, University of Pittsburgh, 3Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 4Infectious Diseases, Department of Pediatrics, Hospital for Sick Children

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

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Medicine

Blind Endotracheal Intubation in Neonatal Rabbits
Sergio Benito 1, Stephanie Hadley 2, Marta Camprubí-Camprubí 3, Joan Sanchez-de-Toledo 4,5
1Department of Pediatric Critical Care, Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, 2Vanderbilt University School of Medicine, 3Neonatology Department, Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, Universidad de Barcelona, 4Department of Pediatric Cardiology, Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, 5Department of Critical Care Medicine, University of Pittsburgh

We describe a technique of endotracheal intubation in newborn rabbits after esophageal catheterization with a gastric tube.

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Cancer Research

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis
Huda I. Atiya 1, Taylor J. Orellana 2, Alyssa Wield 2, Leonard Frisbie 1, Lan G. Coffman 1,2
1Division of Hematology/Oncology, Department of Medicine, Hillman Cancer Center, University of Pittsburgh, 2Division of Gynecologic Oncology, Department of Obstetrics, Gynecology, and Reproductive Sciences, Magee-Womens’ Hospital, University of Pittsburgh Medical Center

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

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Biology

Isolation of Human Primary Valve Cells for In vitro Disease Modeling
Rolando A. Cuevas 1, Claire C. Chu 1, William J. Moorhead III 1, Ryan Wong 1, Ibrahim Sultan 2, Cynthia St. Hilaire 1,3
1Division of Cardiology, Department of Medicine, and the Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, 2Division of Cardiac Surgery, Department of Cardiothoracic Surgery, University of Pittsburgh and Heart and Vascular Institute, University of Pittsburgh Medical Center, 3Department of Bioengineering, University of Pittsburgh

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

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Developmental Biology

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells
Aneta Przepiorski 1, Amanda E. Crunk 1, Teresa M. Holm 2, Veronika Sander 2, Alan J. Davidson 2, Neil A. Hukriede 1,3
1Department of Developmental Biology, University of Pittsburgh, School of Medicine, 2Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, 3Center for Critical Care Nephrology, University of Pittsburgh, School of Medicine

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

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Medicine

Collection, Processing, and Storage Consideration for Urinary Biomarker Research
Nina N. Kowalewski 1, Catherine S. Forster 1
1Department of Pediatrics, University of Pittsburgh

This protocol aims to provide considerations for urine sample collection, processing, and storage for urine tract infection biomarker studies.

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Engineering

Measurement of the Hand Transmitted Vibration of the Human Hand Arm System During Operation of a Hand Tractor
Shiqing Lu 1, Rui Jiang 2, Xinyi Xiao 3, Yong Li 1, Xia Huang 1, Kun Song 1, Changming Chen 1, Jun Ding 1
1School of Mechanical Engineering, Chongqing University of Technology, 2College of Mechanical and Electrical Engineering, Wenzhou University, 3Mechanical and Manufacturing Engineering, Miami University

Here, we present a standardized method for measurement of the hand transmitted vibration from handles of a single-axle tractor with special reference to changes in grip force and vibration frequency.

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Cancer Research

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research
Giovanna Azzarito 1, Marta Ewa Szutkowska 1, Annalisa Saltari 2, Edwin K. Jackson 3, Brigitte Leeners 1, Marinella Rosselli 1, Raghvendra K. Dubey 1,3
1Department of Reproductive Endocrinology, Wagistrasse 14, University of Zurich and University Hospital Zurich, 2Department of Dermatology, Wagistrasse 18, University of Zurich and University Hospital Zurich, 3Department of Pharmacology and Chemical Biology, University of Pittsburgh

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

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Cancer Research

Sentinel Lymph Node Mapping and Biopsy for Endometrial Cancer at Early Stage with Laparoscopy
Bo Wang 1, Yu Xue 1, Qian Wang 1, Yan Xu 1, Xiaojun Chen 1, Chao Wang 1
1Obstetrics and Gynecology Hospital of Fudan University

This protocol describes the identification and resection of sentinel lymph nodes to make the operation as easy and minimally invasive as possible.

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Neuroscience

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats
Matthew T. Rich 1,2, Yanhua H. Huang 1, Mary M. Torregrossa 1
1Department of Psychiatry, University of Pittsburgh, 2Department of Psychiatry, Rutgers University

The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking.

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Biology

Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease
Santana M. Lardo 1, Sarah J. Hainer 1
1Department of Biological Sciences, University of Pittsburgh

CUT&RUN and its variants can be used to determine protein occupancy on chromatin. This protocol describes how to determine protein localization on chromatin using single-cell uliCUT&RUN.

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Cancer Research

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis
Yusuf Surucu *1, Fuat Baris Bengur *1, Katherine S. Yang 1, Benjamin K. Schilling 1, Jocelyn S. Baker 1, Sheeba Shabbir 2, Renee Fisher 3, Michael W. Epperly 3, Joel S. Greenberger 3, J. Peter Rubin 1, Asim Ejaz 1
1Department of Plastic Surgery, University of Pittsburgh, 2Department of Forensic Medicine and Toxicology, HBS Medical and Dental College, 3Department of Radiation Oncology, University of Pittsburgh Cancer Institute

Here we present a protocol to induce radiation-induced skin fibrosis in the hind limb of mice and perform post-irradiation measurements of chronic impairment via limb excursion and gait index analyses to evaluate the functional outcome. The model elucidates radiation-related skin fibrosis mechanisms and is useful in subclinical therapeutic studies.

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Biology

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion
Nikolaos L. Dedousis 1, Lihong Teng 1, Alison B. Kohan 1
1Division of Endocrinology and Metabolism, Department of Medicine, School of Medicine, University of Pittsburgh

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

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Medicine

Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction
Ebin Johny 1, Partha Dutta 1,2,3
1Pittsburgh Heart, Lung, Blood, and Vascular Medicine Institute, Department of Medicine, University of Pittsburgh, 2Department of Immunology, University of Pittsburgh, 3Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh

Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction.

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Medicine

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells
Marcelo D. Carattino 1,2, Wily G. Ruiz 1, Gerard Apodaca 1,2
1Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, 2Department of Cell Biology, University of Pittsburgh

This protocol describes a methodology to assess the function of mechanically activated ion channels in native urothelial cells using the fluorescent Ca2+ sensor GCaMP5G.

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Neuroscience

Pupillometry to Assess Auditory Sensation in Guinea Pigs
Marianny Pernia 1,2, Manaswini Kar 1,2,4, Pilar Montes-Lourido 1,2,6, Srivatsun Sadagopan 1,2,3,4,5
1Department of Neurobiology, University of Pittsburgh, 2Center for Neuroscience, University of Pittsburgh, 3Department of Bioengineering, University of Pittsburgh, 4Center for Neural Basis of Cognition, University of Pittsburgh, 5Department of Communication Science and Disorders, University of Pittsburgh, 6Department of Transfer and Innovation, USC University Hospital Complex (CHUS), University of Santiago de Compostela

Pupillometry, a simple and non-invasive technique, is proposed as a method to determine hearing-in-noise thresholds in normal hearing animals and animal models of various auditory pathologies.

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Developmental Biology

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse
Carla Guzman-Moreno *1, Peizhao Zhang *2, Olivia R. Phillips *1, Mathias Block *3, Benjamin J. Glennon *1, Meghan Holbrook 2, Lauren Weigand 2, Cecilia W. Lo 1, Jiuann-Huey I. Lin 1,4
1Department of Developmental Biology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh, 3Département de Biologie, École Normale Supérieure de Lyon, 4Department of Critical Care Medicine, University of Pittsburgh

This article details murine congenital heart disease (CHD) diagnostic methods using fetal echocardiography, necropsy, and Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) followed by three-dimensional (3D) reconstruction.

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Biology

Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction
Wily G. Ruiz 1, Dennis R. Clayton 1, Marianela G. Dalghi 1, Nicolas Montalbetti 1, Marcelo D. Carattino 1, Gerard Apodaca 1
1Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine

Methods are described for the generation of large amounts of recombinant adenoviruses, which can then be used to transduce the native rodent urothelium allowing for expression of transgenes or downregulation of endogenous gene products.

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Behavior

Real-Time Void Spot Assay
Marianela G. Dalghi 1, Nicolas Montalbetti 1, Travis B. Wheeler 2, Gerard Apodaca 1, Marcelo D. Carattino 1,2
1Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, 2Department of Cell Biology, University of Pittsburgh

This article describes a new method to study mouse voiding behavior by incorporating video monitoring in the conventional void spot assay. This approach provides temporal, spatial, and volumetric information on the voiding events and details of mouse behavior during the light and dark phases of the day.

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Neuroscience

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis
Alisa A. Omelchenko 1,2, Hua Bai 1, Sibtain Hussain 1, Jordan J. Tyrrell 1,3, Mason Klein 4, Lina Ni 1
1School of Neuroscience, Virginia Polytechnic Institute and State University, 2CMU-Pitt Joint Computational Biology, School of Medicine, University of Pittsburgh, 3Eastern Virginia Medical School, 4Department of Physics, University of Miami

TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes.

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Medicine

Pediatric Animal Model of Extracorporeal Cardiopulmonary Resuscitation After Prolonged Circulatory Arrest
Madeleine Ball *1,2, Sergio Benito *1,3, Juliana Pilar Caride 1, Cristina Ruiz-Herguido 1, Marta Camprubí-Camprubí 1,4, Joan Sanchez-de-Toledo 1,5,6
1Sant Joan de Déu Research Institute, 2Vanderbilt University School of Medicine, Vanderbilt University, 3Department of Pediatric Critical Care Medicine, Sant Joan de Déu Hospital, 4BCNatal-Barcelona Center for Maternal Fetal and Neonatal Medicine, Sant Joan de Déu Hospital, 5Department of Pediatric Cardiology, Sant Joan de Déu Hospital, 6Department of Critical Care Medicine, University of Pittsburgh

This protocol describes a neonatal porcine model of cardiopulmonary bypass (CPB), with circulatory and cardiac arrest as a tool for studying severe brain damage and other complications secondary to CPB.

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Neuroscience

Measuring Constipation in a Drosophila Model of Parkinson's Disease
Maggie J. Sodders 1, Ming Shen 1, Abby L. Olsen 1
1Pittsburgh Institute for Neurodegenerative Diseases, Department of Neurology, University of Pittsburgh

This protocol presents an assay for modeling constipation in an alpha-synuclein based Drosophila model of Parkinson's disease.

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Neuroscience

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion
Jeremy Y. Gedeon 1,2,3, Jorge Baruch Pineda-Farias 2,3, Michael S. Gold 2,3
1Center for Neuroscience at the University of Pittsburgh, 2Department of Neurobiology, University of Pittsburgh School of Medicine, 3Pittsburgh Center for Pain Research, University of Pittsburgh

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

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Biology

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing
Line O. Elingaard-Larsen 1,2, Katie L. Whytock 1, Adeline Divoux 1, Meghan Hopf 1, Erin E. Kershaw 3, Jamie N. Justice 4, Bret H. Goodpaster 1, Nancy E. Lane 5, Lauren M. Sparks 1
1Translational Research Institute, AdventHealth, 2Steno Diabetes Center Copenhagen, 3Division of Endocrinology and Metabolism, University of Pittsburgh, 4Gerontology and Geriatric Medicine, Wake Forest University School of Medicine, 5Department of Internal Medicine - Rheumatology, Allergy, and Clinical Immunology, University of California Davis Health

The biology of intermuscular adipose tissue (IMAT) is largely unexplored due to the limited accessibility of human tissue. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing to identify the cellular composition of this unique adipose depot.

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Developmental Biology

Methods to Enable Spatial Transcriptomics of Bone Tissues
Luigi Mancinelli 1,2, Karen Elizabeth Schoedel 3,4, Kurt Richard Weiss 5,6,7, Giuseppe Intini 1,2,8,9,10
1Department of Periodontics and Preventive Dentistry, University of Pittsburgh School of Dental Medicine, 2Center for Craniofacial Regeneration, University of Pittsburgh School of Dental Medicine, 3UPMC Presbyterian, 4UPMC Shadyside, 5Musculoskeletal Oncology Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, 6UPMC Hillman Cancer Center, 7Departments of Anatomic Pathology and General Surgical Oncology, University of Pittsburgh, 8Department of Medicine, University of Pittsburgh School of Medicine, 9University of Pittsburgh UPMC Hillman Cancer Center, 10McGowan Institute for Regenerative Medicine, University of Pittsburgh

Here, we describe a method that allows for the decalcification of freshly obtained bone tissues and the preservation of high-quality RNA. A method is also illustrated for sectioning Formalin Fixed Paraffin Embedded (FFPE) samples of non-demineralized bones to obtain good quality results if fresh tissues are not available or cannot be collected.

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Biology

Studying Orthodontic Tooth Movement in Mice
José Alcides Almeida de Arruda 1, João Pacheco Colares 2, Mariana de Souza Santos 3, Victor Zanetti Drumond 4, Talita Martins 5, Carolina Bosso André 2, Flávio Almeida Amaral 6, Ildeu Andrade Jr. 7, Tarcília Aparecida Silva 8, Soraia Macari 2
1Department of Oral Diagnosis and Pathology, School of Dentistry, Universidade Federal do Rio de Janeiro, 2Department of Restorative Dentistry, School of Dentistry, Universidade Federal de Minas Gerais, 3Department of Physiology and Biophysics, Biological Science Institute, Universidade Federal de Minas Gerais, 4Private Clinic, 5Department of Metallurgical and Materials Engineering, School of Engineering, Universidade Federal de Minas Gerais, 6Laboratory of Immunopharmacology, Department of Biochemistry and Immunology, Biological Science Institute, Universidade Federal de Minas Gerais, 7Department of Orthodontics, School of Dental Medicine, University of Pittsburgh, 8Department of Oral Surgery, Pathology and Clinical Dentistry, School of Dentistry, Universidade Federal de Minas Gerais

Here, we present a protocol for studying orthodontic tooth movement (OTM), serving as a suitable model for investigating the mechanisms of bone adaptation, root resorption, and the response of bone cells to mechanical stimuli. This comprehensive guide provides detailed information on the OTM model, micro-computed tomography acquisition, and subsequent analysis.

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Neuroscience

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis
Nadezda A. Stepicheva 1, Zhongfang Weng 1,2, Sarah Cao 3, Lesley M. Foley 4, Guodong Cao 1,2
1Department of Neurology, University of Pittsburgh, 2Geriatric Research Education and Clinical Center, VA Pittsburgh Healthcare System, 3School of Arts & Sciences, Washington University in St. Louis, 4Animal Imaging Center, University of Pittsburgh

The needle method of asymmetric bilateral common carotid artery stenosis is proposed to create a mouse model for vascular cognitive impairment and dementia. It results in longer-term outcomes compared to previously established models and is compatible with live MRI. Visual representation demonstrating the procedure provides guidance for mastering the surgery.

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Medicine

Microfluidics in Assessing Platelet Function
Emily Mihalko 1, Abiha Abdullah 1, Lara Hoteit 1, David Akinbode 1, Susan Shea 1, Matthew D. Neal 1
1Trauma and Transfusion Medicine Research Center, Department of Surgery, University of Pittsburgh

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

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