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85 ARTICLES PUBLISHED IN JoVE

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Biology

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging
Melissa Paulite 1, Zahra Fakhraai 2, Boris B. Akhremitchev 3, Kerstin Mueller 1, Gilbert C. Walker 1
1Department of Chemistry, University of Toronto, 2Department of Chemistry, University of Wisconsin, 3Department of Chemistry, Duke University

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

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Biology

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture
Jamie L. Ifkovits 1, Harini G. Sundararaghavan 1, Jason A. Burdick 1
1Department of Bioengineering, University of Pennsylvania

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

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Neuroscience

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence
Sava Sakadžić 1, Emmanuel Roussakis 2, Mohammad A. Yaseen 1, Emiri T. Mandeville 3, Vivek J. Srinivasan 1, Ken Arai 3, Svetlana Ruvinskaya 1, Weicheng Wu 1, Anna Devor 1,4, Eng H. Lo 3, Sergei A. Vinogradov 2, David A. Boas 1
1Optics Division, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2Department of Biochemistry and Biophysics, University of Pennsylvania, 3Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, 4Departments of Neurosciences and Radiology, University of California

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

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Biology

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems
Louise E. Sinks 1, Emmanuel Roussakis 1, Tatiana V. Esipova 1, Sergei A. Vinogradov 1
1Department of Biochemistry and Biophysics, University of Pennsylvania

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

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Biology

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans
Marni J. Falk 1,2, Meera Rao *1, Julian Ostrovsky *1, Evgueni Daikhin 1, Ilana Nissim 1, Marc Yudkoff 1,2
1Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

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Biology

Crystallization of Membrane Proteins in Lipidic Mesophases
Wei Liu 1, Vadim Cherezov 1
1Molecular Biology, The Scripps Research Institute

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

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Neuroscience

Transcriptome Analysis of Single Cells
Jacqueline Morris *1, Jennifer M. Singh *1, James H. Eberwine 1,2
1Department of Pharmacology, University of Pennsylvania, 2The Penn Genome Frontiers Institute, University of Pennsylvania

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

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Biology

In vitro Reconstitution of the Active T. castaneum Telomerase
Anthony P. Schuller 1, Michael J. Harkisheimer 1, Emmanuel Skordalakes 1
1Gene Expression and Regulation, The Wistar Institute, University of Pennsylvania

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

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Biology

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment
Paula Stein 1, Karen Schindler 1
1Department of Biology, University of Pennsylvania

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

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Biology

Purification of Hsp104, a Protein Disaggregase
Elizabeth A. Sweeny 1, Morgan E. DeSantis 1, James Shorter 1
1Department of Biochemistry and Biophysics, University of Pennsylvania

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

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Bioengineering

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model
Alexandra E. Jantzen 1, Whitney O. Lane 2, Shawn M. Gage 3, Justin M. Haseltine 1, Lauren J. Galinat 1, Ryan M. Jamiolkowski 4, Fu-Hsiung Lin 3, George A. Truskey 1, Hardean E. Achneck 3
1Department of Biomedical Engineering, Duke University , 2School of Medicine, Duke University , 3Department of Surgery, Duke University Medical Center, 4School of Medicine, University of Pennsylvania

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

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Bioengineering

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress
Whitney O. Lane 1, Alexandra E. Jantzen 2, Tim A. Carlon 2, Ryan M. Jamiolkowski 3, Justin E. Grenet 1, Melissa M. Ley 1, Justin M. Haseltine 2, Lauren J. Galinat 2, Fu-Hsiung Lin 1, Jason D. Allen 4, George A. Truskey 2, Hardean E. Achneck 1
1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University , 3School of Medicine, University of Pennsylvania , 4Department of Medicine, Division of Cardiology, Duke University Medical Center

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

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Bioengineering

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
Stacy L. Gelhaus 1,2, A. Clementina Mesaros 1,2, Ian A. Blair 1,2
1Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania , 2Department of Pharmacology, University of Pennsylvania

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

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Immunology and Infection

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model
Dimitrios N. Vatakis 1,2,3, Gregory C. Bristol 1,2, Sohn G. Kim 1,2, Bernard Levin 1,2, Wei Liu 4, Caius G. Radu 4, Scott G. Kitchen 1,2,3, Jerome A. Zack 1,2,5
1Department of Medicine, Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, 2UCLA AIDS Institute, 3Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, 4Department of Medical and Molecular Pharmacology, David Geffen School of Medicine at UCLA, 5Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

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Biology

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector
Andreia Gianotti Sommer 1, Sarah S. Rozelle 1, Spencer Sullivan 2, Jason A. Mills 3, Seon-Mi Park 1, Brenden W. Smith 1, Amulya M. Iyer 1, Deborah L. French 3, Darrell N. Kotton 1, Paul Gadue 3, George J. Murphy 1, Gustavo Mostoslavsky 1
1Center for Regenerative Medicine (CReM), Boston University School of Medicine, 2Department of Hematology, Children's Hospital of Philadelphia, 3Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

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Medicine

Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia
Gabriella Garcia 1,2, Catherine Norise 1,2, Olufunsho Faseyitan 1,2, Margaret A. Naeser 3,4,5, Roy H. Hamilton 1,2
1Department of Neurology, Perelman School of Medicine, University of Pennsylvania , 2Center for Cognitive Neuroscience, University of Pennsylvania , 3Veterans Affairs Boston Healthcare System, 4Harold Goodglass Aphasia Research Center, Boston University School of Medicine, 5Department of Neurology, Boston University School of Medicine

We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.

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Chemistry

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)
Nathaniel W. Snyder 1, Maya Khezam 1, Clementina A. Mesaros 1, Andrew Worth 1, Ian A. Blair 1
1Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

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Biology

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation
Jessica M Bryant 1,2, Mirella L Meyer-Ficca 3, Vanessa M Dang 1, Shelley L Berger 1,4,5, Ralph G Meyer 3
1Department of Cell and Developmental Biology, University of Pennsylvania, 2Biomedical Graduate Studies, University of Pennsylvania, 3Department of Animal Biology and Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, 4Department of Genetics, University of Pennsylvania, 5Department of Biology, University of Pennsylvania

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

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Neuroscience

A Single-fly Assay for Foraging Behavior in Drosophila
Orel A. Zaninovich 1, Susy M. Kim 1, Cory R. Root 1,2, David S. Green 1,3, Kang I. Ko 1,4, Jing W. Wang 1
1Neurobiology Section, Division of Biological Sciences, University of California-San Diego, 2Department of Neuroscience, Columbia University, 3Dart NeuroScience, 4School of Dental Medicine, University of Pennsylvania

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

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JoVE Core

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention
Hilary S. McCarren 1,2,4, Jason T. Moore 1,3,4, Max B. Kelz 1,2,3,4
1Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, 2Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, 3Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, 4Center for Sleep and Circadian Neurobiology, Perelman School of Medicine, University of Pennsylvania

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

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Biology

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends
Vladimir A. Volkov 1,2, Anatoly V. Zaytsev 3, Ekaterina L. Grishchuk 3
1Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, 2Federal Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia, 3Physiology Department, Perelman School of Medicine, University of Pennsylvania

Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.

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Medicine

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer
Melanie R. Rutkowski *1, Michael J. Allegrezza *1, Nikolaos Svoronos 1, Amelia J. Tesone 1, Tom L. Stephen 1, Alfredo Perales-Puchalt 1, Jenny Nguyen 1, Paul J. Zhang 2, Steven N. Fiering 3, Julia Tchou 4,5,6, Jose R. Conejo-Garcia 1
1Tumor Microenvironment and Metastasis Program, Wistar Institute, 2Department of Pathology and Lab Medicine, Perelman School of Medicine, University of Pennsylvania, 3Department of Microbiology and Immunology and Department of Genetics, Geisel School of Medicine at Dartmouth, 4Division of Endocrine and Oncologic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, 5Rena Rowan Breast Center, Abramson Cancer Center, University of Pennsylvania, 6Center for Advanced Medicine, University of Pennsylvania

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

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Neuroscience

Using Informational Connectivity to Measure the Synchronous Emergence of fMRI Multi-voxel Information Across Time
Marc N. Coutanche 1, Sharon L. Thompson-Schill 1
1Department of Psychology, University of Pennsylvania

Informational connectivity measures the correspondence between time courses of multi-voxel information across different brain regions. Multi-voxel pattern discriminability time series are extracted from regions and compared, revealing networks that are not identified in a typical functional connectivity approach.

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Bioengineering

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration
Lindsay M. Thomson 1, Brian D. Polizzotti 1, Frances X. McGowan 2, John N. Kheir 1
1Department of Cardiology, Boston Children's Hospital, Harvard Medical School, 2Department of Anesthesiology and Critical Care, Children's Hospital of Philadelphia, University of Pennsylvania

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

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Biology

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
Yang Song *1, Xuemei Zhang *1, Lingyan Huang 2, Mark A. Behlke 2, Andrew Tsourkas 1
1Department of Bioengineering, University of Pennsylvania, 2Integrated DNA Technologies, Inc.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

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Medicine

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers
Ilia Fishbein 1, Scott P. Forbes 1, Richard F. Adamo 1, Michael Chorny 1, Robert J. Levy 1, Ivan S. Alferiev 1
1Department of Pediatrics, Division of Cardiology, The Children's Hospital of Philadelphia, University of Pennsylvania

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

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Bioengineering

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits
Joshua B. Slee 1,2, Ivan S. Alferiev 1,2, Robert J. Levy 1,2, Stanley J. Stachelek 1,2
1Division of Cardiology, Department of Pediatrics, The Children's Hospital of Philadelphia, 2University of Pennsylvania Perelman School of Medicine

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

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Biology

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models
Meredith E. Jackrel 1, Amber Tariq 1, Keolamau Yee 1, Rachel Weitzman 1, James Shorter 1
1Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

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Bioengineering

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities
Jacob R. Price 1, Wen K. Shieh 2, Christopher M. Sales 1
1Civil, Architectural, and Environmental Engineering, Drexel University, 2Chemical and Biomolecular Engineering, University of Pennsylvania

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

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Chemistry

Cooling Rate Dependent Ellipsometry Measurements to Determine the Dynamics of Thin Glassy Films
Ethan C. Glor 1, Zahra Fakhraai 1
1Department of Chemistry, University of Pennsylvania

Here, we present a protocol for cooling rate dependent ellipsometry experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glass for a variety of glassy materials.

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Education

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism
Andrew J. Worth *1,2, Dylan M. Marchione *2,3, Robert C. Parry 1,2, Qingqing Wang 2,3, Kevin P. Gillespie 2,3, Noelle N. Saillant 4, Carrie Sims 4, Clementina Mesaros 1,2, Nathaniel W. Snyder 5, Ian A. Blair 1,2
1Center for Cancer Pharmacology, University of Pennsylvania, 2Center for Excellence in Environmental Toxicology, University of Pennsylvania, 3Penn SRP and Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, 4Division of Traumatology, Department of Surgery, Critical Care and Acute Care Surgery, University of Pennsylvania, 5A.J. Drexel Autism Institute, Drexel University

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

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Medicine

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes
Gregory P. Way 1,2, Maura Southwell 1, Scott P. McRobert 1
1Department of Biology, Saint Joseph’s University, 2Genomics and Computational Biology, University of Pennsylvania

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

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Biology

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis
Simone Sidoli 1, Natarajan V. Bhanu 1, Kelly R. Karch 1, Xiaoshi Wang 1, Benjamin A. Garcia 1
1Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

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Biochemistry

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography
Andrii Ishchenko 1,2, Vadim Cherezov 1,2, Wei Liu 3
1The Bridge Institute, University of Southern California, 2Department of Chemistry, University of Southern California, 3School of Molecular Sciences, Center for Applied Structural Discovery at the Biodesign Institute, Arizona State University

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

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Biology

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy
Yong Ho Bae 1,2, Shu-lin Liu 1,2, Fitzroy J. Byfield 3, Paul A. Janmey 3, Richard K. Assoian 1,2
1Program in Translational Biomechanics, Institute of Translational Medicine and Therapeutics, University of Pennsylvania, 2Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, 3Department of Physiology, University of Pennsylvania

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

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Environment

Using Pharmacological Manipulation and High-precision Radio Telemetry to Study the Spatial Cognition in Free-ranging Animals
Timothy C. Roth *1, Aaron R. Krochmal *2, William B. Gerwig, IV 1, Sage Rush 3, Nathaniel T. Simmons 2, Jeffery D. Sullivan 4, Katrina Wachter 5
1Department of Psychology, Franklin and Marshall College, 2Department of Biology, Washington College, 3University of Pennsylvania, 4School of Forestry and Wildlife Sciences, Auburn University, 5Morsani College of Medicine, University of South Florida

This paper describes a novel protocol that combines the pharmacological manipulation of memory and radio telemetry to document and quantify the role of cognition in navigation.

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Neuroscience

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources
Andrzej Z. Wasilczuk 1, Alexander Proekt 1, Max B. Kelz 1, Andrew R. McKinstry-Wu 1
1Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

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Medicine

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle
Vidhya Kumar 1, Henry Chang 1, David A. Reiter 2, David P. Bradley 3, Martha Belury 4, Shana E. McCormack 5, Subha V. Raman 1
1Davis Heart and Lung Research Institute, The Ohio State University, 2Laboratory of Clinical Investigation, National Institute on Aging, 3Division of Endocrinology, Diabetes and Metabolism, The Ohio State University, 4Department of Human Sciences, Human Nutrition, The Ohio State University, 5Division of Endocrinology and Diabetes, Department of Pediatrics, University of Pennsylvania

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

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JoVE Core

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells
Jianle Wang 1, Montserrat C. Anguera 1
1Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

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Neuroscience

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling
Laura A. Struzyna *1,2,3, Dayo O. Adewole *1,2,3, Wisberty J. Gordián-Vélez 1,2,3, Michael R. Grovola 2,3, Justin C. Burrell 2,3, Kritika S. Katiyar 2,3,4, Dmitriy Petrov 2,3, James P. Harris 2,3, D. Kacy Cullen 2,3
1Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, 2Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, 3Center for Neurotrauma, Neurodegeneration & Restoration, Michael J. Crescenz Veterans Affairs Medical Center, 4School of Biomedical Engineering, Drexel University

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

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JoVE Journal

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration
Kritika S. Katiyar *1,2,3, Carla C. Winter *1,2,4, Wisberty J. Gordián-Vélez 1,2,4, John C. O'Donnell 1,2, Yeri J. Song 1,5, Nicole S. Hernandez 1,5, Laura A. Struzyna 1,2,4, D. Kacy Cullen 1,2,5
1Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, 2Center for Neurotrauma, Neurodegeneration & Restoration, Michael J. Crescenz Veterans Affairs Medical Center, 3School of Biomedical Engineering, Drexel University, 4Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, 5Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered "living scaffolds", exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

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Bioengineering

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening
Yaqian Li *1,2, Xiaojun Yan *1, Wei Liu *1, Lyu Zhou 1,3, Zhifeng You 1, Yanan Du 1,2
1Department of Biomedical Engineering, School of Medicine, Tsinghua University, 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, 3School of Life Sciences, Tsinghua University

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

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Biochemistry

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
Robert Warneford-Thomson 1,4, Chongsheng He 1,2, Simone Sidoli 1,3, Benjamin A. Garcia 1,3, Roberto Bonasio 1,2
1Epigenetics Institute, University of Pennsylvania Perelman School of Medicine, 2Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 3Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, 4Graduate Group in Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

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Medicine

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue
Emanuele Loro *1, Stephen H. Wang *2, Richard J. Schwab 2, Tejvir S. Khurana 1
1Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, 2Center for Sleep & Circadian Neurobiology, University of Pennsylvania

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

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Neuroscience

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors
David J. Tischfield 1,2, Stewart A. Anderson 1
1Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, 2Department of Psychiatry, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

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Bioengineering

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis
Yan Li *1, Hannah Grover *1, Eric Dai 2, Kevin Yang 1, Zi Chen 1
1Thayer School of Engineering, Dartmouth College, 2Department of Bioengineering, University of Pennsylvania

Here, we present a protocol introducing a set of new ex-ovo experiments and physical modeling approaches for studying the mechanics of morphogenesis during early chick embryonic brain torsion.

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Education

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I
Diego Marin *1,2, Alexandra L. Nguyen *3, Richard T. Scott, Jr. 1,2, Karen Schindler 3
1IVI-RMA New Jersey, 2Jefferson College of Biomedical Sciences, Thomas Jefferson University, 3Department of Genetics, Rutgers, The State University of New Jersey

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

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JoVE Journal

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System
Dan Li *1, Feng Li *1, Pavithran Guttipatti 1, Yuanquan Song 1,2
1Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, 2Department of Pathology and Laboratory Medicine, University of Pennsylvania

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

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Genetics

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms
Clark Fritsch 1,2, Jean-Francois Pierre Gout 3,4, Marc Vermulst 1
1Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, 2Department of Cellular and Molecular Biology, University of Pennsylvania, 3Department of Biological Sciences, Mississippi State University, 4Center for Mechanisms of Evolution, Biodesign Institute, Arizona State University

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

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Using Dynamic, Interactive Paradigms to Study Social Neuroscience in Rhesus Macaque Monkeys
Yaoguang Jiang 1, Michael L Platt 1,2,3
1Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, 2Department of Psychology, School of Arts and Sciences, University of Pennsylvania, 3Marketing Department, the Wharton School, University of Pennsylvania

Here we present two protocols to examine the neuronal mechanisms underlying live interactions between pairs of rhesus macaque monkeys, in an effort to gain insight into the neurobiology of complex social behaviors in humans.

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Immunology and Infection

Merkel Cell Polyomavirus Infection and Detection
Wei Liu *1, Nathan A. Krump *1, Christopher B. Buck 2, Jianxin You 1
1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Laboratory of Cellular Oncology, National Cancer Institute

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

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JoVE Journal

Experimental Paradigm for Measuring the Effects of Self-distancing in Young Children
Amanda Grenell 1, Rachel E. White 2, Emily O. Prager 1, Catherine Schaefer 1, Ethan Kross 3, Angela L. Duckworth 4, Stephanie M. Carlson 1
1Insitute of Child Development, University of Minnesota-Twin Cities, 2Department of Psychology, Hamilton College, 3Department of Psychology, University of Michigan, 4Department of Psychology, University of Pennsylvania

An experimental paradigm was created to measure the effects of self-distancing in young children (4-6-year-olds). Self-distancing is a process through which individuals adopt a less egocentric perspective. This paradigm has been used to examine the effects of self-distancing on young children's self-regulation.

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Genetics

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
Fabian L. Cardenas-Diaz *1, Jean Ann Maguire *1, Paul Gadue 1,2,3, Deborah L. French 1,2,3
1Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, 2Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, 3Department of Pathology and Laboratory Medicine, University of Pennsylvania

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

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JoVE Journal

Cell Type-specific Gene Expression Profiling in the Mouse Liver
Amber W. Wang 1, Adam M. Zahm 1, Kirk J. Wangensteen 1,2
1Department of Genetics, University of Pennsylvania, 2Department of Medicine, University of Pennsylvania

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

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Cancer Research

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma
Ceire A. Hay 1, Rina Sor 2,3, Ahron J. Flowers 2,3, Cynthia Clendenin 2,3, Katelyn T. Byrne 1,4
1Department of Medicine, Perelman School of Medicine, University of Pennsylvania, 2Pancreatic Cancer Mouse Hospital, Perelman School of Medicine, University of Pennsylvania, 3Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, 4Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

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Bioengineering

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording
Nicolette Driscoll 1,2,3, Kathleen Maleski 4,5, Andrew G. Richardson 2,6, Brendan Murphy 1,2,3, Babak Anasori 4,5, Timothy H. Lucas 2,6, Yury Gogotsi 4,5, Flavia Vitale 1,2,3,7,8
1Department of Bioengineering, University of Pennsylvania, 2Center for Neuroengineering and Therapeutics, University of Pennsylvania, 3Corporal Michael J. Crescenz Veterans Affairs Medical Center, 4Department of Materials Science and Engineering, Drexel University, 5A.J. Drexel Nanomaterials Institute, Drexel University, 6Department of Neurosurgery, University of Pennsylvania, 7Department of Neurology, University of Pennsylvania, 8Department of Physical Medicine and Rehabilitation

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

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Bioengineering

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy
Kevin Y. Chen 1, Ryan M. Jamiolkowski 1, Alyssa M. Tate 1, Shane A. Fiorenza 2, Shawn H. Pfeil 2, Yale E. Goldman 1
1Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, 2Department of Physics, West Chester University

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

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Behavior

Assessing Binocular Central Visual Field and Binocular Eye Movements in a Dichoptic Viewing Condition
Rajkumar N. Raveendran *1, Arun K. Krishnan *1,2
1Envision Research Institute, 2University of Pennsylvania

Presented here is a protocol for assessing binocular eye movements and gaze-controlled central visual field screening in participants with central vision loss.

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Bioengineering

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization
Vaishali V. Inamdar 1, Emmett G. Fitzpatrick 1, Ivan S. Alferiev 1,2, Robert J. Levy 1,2, Stanley J. Stachelek *1,2, Ilia Fishbein *1,2
1The Children's Hospital of Philadelphia, 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

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JoVE Core

Lower-Limb Biomechanical Characteristics Associated with Unplanned Gait Termination Under Different Walking Speeds
Huiyu Zhou 1,2, Xuanzhen Cen 1, Yang Song 3, Ukadike C. Ugbolue 2, Yaodong Gu 1
1Faculty of Sports Science, Ningbo University, 2School of Health and Life Sciences, University of the West of Scotland, 3Faculty of Engineering, University of Szeged

This study compared the biomechanical characteristics of the lower extremity during unplanned gait termination under different walking speeds. The lower-limb kinematic and kinetic data from fifteen subjects with normal and fast walking speeds were collected using a motion analysis system and plantar pressure platform.

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Immunology and Infection

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach
Yasmine Issah 1, Amruta Naik 1, Soon Yew Tang 2,3, Kaitlyn Forrest 1, Katherine N. Theken 2,3, Shaon Sengupta 1,3,4
1The Children's Hospital of Philadelphia, 2Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, 3Institute of Translational Medicine and Therapeutics (ITMAT), University of Pennsylvania, 4Department of Pediatrics, University of Pennsylvania Perelman School of Medicine

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

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Developmental Biology

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad
Kara A. Nelson 1, Bailey N. Warder 1, Stephen DiNardo 1, Lauren Anllo 1
1Cell and Developmental Biology Department and the Penn Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

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Medicine

Virtual Reality Tools for Assessing Unilateral Spatial Neglect: A Novel Opportunity for Data Collection
Peter J. Schwab 1,2, Alex Miller 2, Ann-Marie Raphail 3, Ari Levine 2, Christopher Haslam 2, H. Branch Coslett 1,2, Roy H. Hamilton 1,2,4
1Department of Neurology, University of Pennsylvania, 2Laboratory for Cognition and Neural Stimulation, University of Pennsylvania, 3Department of Psychology, Drexel University, 4Department of Physical Medicine and Rehabilitation, University of Pennsylvania

The goal was to design, build, and pilot a novel virtual reality task to detect and characterize unilateral spatial neglect, a syndrome affecting 23-46% of acute stroke survivors, expanding the role of virtual reality in the study and management of neurologic disease.

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Cancer Research

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate
Shun Ishiyama 1,2,3,4, Casey Kissel 5, Xin Guo 1, Alexis Howard 6, Harumi Saeki 7, Tomoaki Ito 8, Polina Sysa-Shah 9, Hajime Orita 10, Kazuhiro Sakamoto 4, Kathleen Gabrielson 1,2
1Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, 2Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 3Department of Surgery, Johns Hopkins University School of Medicine, 4Department of Coloproctological Surgery, Juntendo University Faculty of Medicine, 5Program for Comparative Medicine, Gene Therapy Program, Perelman School of Medicine, University of Pennsylvania, 6Tuskegee College of Veterinary Medicine, 7Department of Human Pathology, Juntendo University Faculty of Medicine, 8Department of Surgery, Juntendo University Shizuoka Hospital, Juntendo University School of Medicine, 9Department of Urology, The Johns Hopkins School of Medicine, 10Department of Gastroenterology and Minimally Invasive Surgery, Juntendo University Faculty of Medicine

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

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Biology

Chemical Dimerization-Induced Protein Condensates on Telomeres
Rongwei Zhao 1, David M. Chenoweth 2, Huaiying Zhang 1
1Department of Biological Sciences, Mellon College of Science, Carnegie Mellon University, 2Department of Chemistry, School of Arts and Sciences, University of Pennsylvania

This protocol illustrates a chemically induced protein dimerization system to create condensates on chromatin.  The formation of promyelocytic leukemia (PML) nuclear body on telomeres with chemical dimerizers is demonstrated. Droplet growth, dissolution, localization and composition are monitored with live cell imaging, immunofluorescence (IF) and fluorescence in situ hybridization (FISH).

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JoVE Journal

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays
Ananya Tripathi 1, Charles Bond 1,2, James R. Sellers 1, Neil Billington 1, Yasuharu Takagi 1
1Laboratory of Molecular Physiology, Cell and Developmental Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, 2Department of Physiology, Perelman School of Medicine, University of Pennsylvania

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

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Behavior

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease
Manuela Lavorato *1, Neal D. Mathew *1, Nina Shah 1, Eiko Nakamaru-Ogiso 1,2, Marni J. Falk 1,2
1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania Perelman School of Medicine

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

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Immunology and Infection

Production of Human CRISPR-Engineered CAR-T Cells
Sangya Agarwal *1,2, Nils Wellhausen *1,2, Bruce L. Levine 1,2, Carl H. June 1,2
1Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, 2Parker Institute for Cancer Immunotherapy, University of Pennsylvania

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

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Biochemistry

Purification of Endogenous Drosophila Transient Receptor Potential Channels
Jia Liu *1, Yuyang Liu *1, Weidi Chen 1, Yuzhen Ding 1, Xiaoru Lan 1, Wei Liu 1
1Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

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Biology

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue
Jazmine-Saskya N. Joseph-Chowdhury 1, Stephanie Stransky 1, Sarah Graff 1, Ronald Cutler 1, Dejauwne Young 1, Julie S. Kim 1, Carlos Madrid-Aliste 1, Jennifer T. Aguilan 1, Edward Nieves 1, Yan Sun 1, Edwin J. Yoo 1, Simone Sidoli 1
1Department of Biochemistry, Albert Einstein College of Medicine

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

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Bioengineering

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications
Victoria G. Muir 1, Margaret E. Prendergast 1, Jason A. Burdick 1,2,3
1Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, 2BioFrontiers Institute, University of Colorado Boulder, 3Department of Chemical and Biological Engineering, College of Engineering and Applied Science, University of Colorado Boulder

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

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Biology

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis
Jinxi Yuan 1, Jie Zhang 1, Yan Zhang 1, WuYun QiQiGe 1, Wei Liu 2, Shanchun Yan 1, Guirong Wang 2
1Key Laboratory of Sustainable Forest Ecosystem Management - Ministry of Education, Northeast Forestry University, 2Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

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Biochemistry

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection
Jonathan T. Specker 1, Alexander B. Smith 2, Orlaith Keenan 2, Joseph P. Zackular 2,3, Boone M. Prentice 1
1Department of Chemistry, University of Florida, 2Division of Protective Immunity, Children’s Hospital of Philadelphia, 3Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

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Developmental Biology

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes
Mansour Aboelenain 1,2,3, Karen Schindler 1,2, Cecilia S. Blengini 1,2
1Department of Genetics, Rutgers, The State University of New Jersey, 2Human Genetics Institute of New Jersey, 3Department of Theriogenology, Faculty of Veterinary Medicine, Mansoura University

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

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Bioengineering

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue
Amanda L. Holt 1,3, Yakir Luc Gagnon 2,4, Alison M. Sweeney 1,3
1Department of Physics, Yale University, 2Lund Vision Group, Department of Biology, Lund University, 3Department of Physics and Astronomy, University of Pennsylvania, 4Department of Biology, Duke University

In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data.

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Biology

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices
Emily A. Gardea 1, Destiny DeNicola 1, Samuel Freitas 1, Will Peterson 1, Hope Dang 1, Karissa Shuck 1, Christopher Fang-Yen 2, George L. Sutphin 1
1Department of Molecular & Cellular Biology, University of Arizona, 2Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

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Bioengineering

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing
Rakan El-Mayta 1, Marshall S. Padilla 1, Margaret M. Billingsley 1, Xuexiang Han 1, Michael J. Mitchell 1
1Department of Bioengineering, University of Pennsylvania

Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice.

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Immunology and Infection

Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions
Clayton J. Otter 1,2, Alejandra Fausto 1,2, Li Hui Tan 3,4, Susan R. Weiss 1,2, Noam A. Cohen 3,4
1Department of Microbiology, University of Pennsylvania, 2Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, 3Otorhinolaryngology-Head and Neck Surgery, University of Pennsylvania, 4Corporal Michael J. Crescenz VA Medical Center

The nasal epithelium is the primary barrier site encountered by all respiratory pathogens. Here, we outline methods to use primary nasal epithelial cells grown as air-liquid interface (ALI) cultures to characterize human coronavirus-host interactions in a physiologically relevant system.

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Cancer Research

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines
Kyra Harvey 1,2,3, Katherine Labella 1,2,3, Angela Liou 1,2,3, Stephanie Brosius 1,2,3, Thomas De Raedt 1,2,3
1Division of Oncology, Children’s Hospital of Philadelphia, 2Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, 3School of Medicine, University of Pennsylvania

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

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Bioengineering

Finite Element Analysis Model for Assessing Expansion Patterns from Surgically Assisted Rapid Palatal Expansion
Jia-Hong Lin *1, Guan-Lin Wu *2, Chun-Kai Chiu 2, Steven Wang 3, Chun-Hsi Chung 1, Chenshuang Li 1
1Department of Orthodontics, School of Dental Medicine, University of Pennsylvania, 2Department of Biomedical Engineering, College of Engineering, National Cheng Kung University, 3Department of Oral and Maxillofacial Surgery/Pharmacology, School of Dental Medicine, University of Pennsylvania

A set of novel finite element models of surgically assisted rapid palatal expansion (SARPE) that could perform a clinically required amount of expander activation with various angles of buccal osteotomy was created for further analysis of the expansion patterns of the hemimaxillae in all three dimensions.

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Behavior

Tracking Sugar-Elicited Local Searching Behavior in Drosophila
Manal Shakeel 1,2, Shivam Kaushik 3, Teiichi Tanimura 4, Axel Brockmann 1, Pinky Kain 3,5
1National Centre for Biological Sciences, 2The University of Trans-Disciplinary Health Sciences and Technology, 3Regional Centre for Biotechnology, 4Neural Circuit Group, Division of Biological Science, Graduate School of Science, Nagoya University, 5Perelman School of Medicine, University of Pennsylvania

This protocol describes a behavioral assay for recording sugar-elicited search behavior using Drosophila melanogaster. The assay can be utilized to study feeding and foraging-related behaviors, as well as the underlying neuronal mechanisms.

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Neuroscience

The Sol Braiding Method for Handling Thick Hair During Transcranial Magnetic Stimulation: An Address for Potential Bias in Brain Stimulation
Qiana Archer 1, Janet Brenya 1, Katherine Chavaria 2, Anjel Friest 3, Nathira Ahmad 4, Samantha Zorns 1, Sahana Vaidya 1, Taylor Shelanskey 1, Sarah Sierra 1, Sydney Ash 1, Briana Balugus 5, Alexa Alvarez 6, Mathew Pardillo 1, Roy Hamilton 7, Julian Paul Keenan 1
1Cognitive Neuroimaging Laboratory, Montclair State University, 2School of Osteopathic Medicine, Rowan University, 3Department of Biology, Rutgers University, 4Department of Health Informatics, Rutgers University, 5Department of Physician Assistant, Seton Hall University, 6School of Nursing, Felician College, 7Laboratory for Cognition and Neural Stimulation, University of Pennsylvania

Hair type commonly seen in historically underrepresented minorities appears to interfere with transcranial magnetic stimulation (TMS). Here we describe a hair braiding method (The Sol Braiding Technique) that improves TMS.

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Medicine

Quantitative Measure of Lung Structure and Function Obtained from Hyperpolarized Xenon Spectroscopy
Kai Ruppert 1, Luis Loza 1, Faraz Amzajerdian 1, Hooman Hamedani 1, Ryan Baron 1, Stephen Kadlecek 1, Rahim Rizi 1
1Department of Radiology, University of Pennsylvania

The manuscript presents a detailed protocol for using hyperpolarized Xenon-129 chemical shift saturation recovery (CSSR) to trace pulmonary gas exchange, assess the apparent alveolar septal wall thickness, and measure the surface-to-volume ratio. The method has the potential to diagnose and monitor lung diseases.

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Cancer Research

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models
Beatriz Goncalves 1, Shujing Liu 1, Xiaogang Zhang 1, Andrew Fan 1, Lingling Ou 1, Xiaowei Xu 1
1Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania

Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches.

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Biology

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy
Irene Santiago Tierno 1,2,3, Mahesh Agarwal 1,2, Nikolaos Matisioudis 2,4, Sathishkumar Chandrakumar 1,2, Kaustabh Ghosh 1,2,3
1Department of Ophthalmology, University of California, Los Angeles, 2Doheny Eye Institute, 3Molecular, Cellular, and Integrative Physiology Interdepartmental PhD Program, University of California, Los Angeles, 4Department of Bioengineering, University of Pennsylvania

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.

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