S'identifier

Boston University

43 ARTICLES PUBLISHED IN JoVE

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Biology

Wolbachia Bacterial Infection in Drosophila
Horacio Frydman 1
1Boston University

Wolbachia Bacterial Infection in Drosophila

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Biology

Fabrication of the Thermoplastic Microfluidic Channels
Arpita Bhattacharyya 1, Dominika Kulinski 1, Catherine Klapperich 1
1Department of Biomedical Engineering, Boston University

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

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Biology

Microfluidic Applications for Disposable Diagnostics
Catherine Klapperich 1
1Department of Biomedical Engineering, Boston University

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

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Biology

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice
Birgit Werner 1, Paul B. Cook 1,2, Christopher L. Passaglia 1,3
1Program in Neuroscience, Boston University, 2Department of Biology, Boston University, 3Department of Biomedical Engineering, Boston University

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

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Biology

Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research
Jiahui S. Liu 1, Christopher L. Passaglia 1
1Department of Biomedical Engineering, Boston University

In this video we perform electroretinogram recording, optic nerve recording, and intraretinal recording with the American horseshoe crab, Limulus Polyphemus. These electrophysiological paradigms can be used for investigating the neural basis of vision in a research or teaching lab.

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Biology

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules
Nicolas Di Fiori 1, Amit Meller 1,2
1Physics Department, Boston University, 2Department of Biomedical Engineering, Boston University

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

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Neuroscience

Single-unit In vivo Recordings from the Optic Chiasm of Rat
Daniel K. Freeman 1, Walter F. Heine 1, Christopher L. Passaglia 1
1Department of Biomedical Engineering, Boston University

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

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Bioengineering

Microfluidic Chip Fabrication and Method to Detect Influenza
Qingqing Cao 1, Andy Fan 2, Catherine Klapperich 1,2
1Department of Mechanical Engineering, Boston University , 2Department of Biomedical Engineering, Boston University

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

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Environment

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)
Jason S Goldstein 1, Tracy L Pugh 1,2, Elizabeth A Dubofsky 1, Kari L Lavalli 3, Michael Clancy 4, Winsor H Watson III 1
1Department of Biological Sciences, University of New Hampshire, 2Massachusetts Division of Marine Fisheries, 3Division of Natural Sciences & Mathematics, College of General Studies, Boston University, 4Rhode Island Nursing Institute, Middle College

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study's goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

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Bioengineering

Stress-induced Antibiotic Susceptibility Testing on a Chip
Maxim Kalashnikov 1, Jennifer Campbell 1, Jean C. Lee 2, Andre Sharon 1,3, Alexis F. Sauer-Budge 1,4
1Fraunhofer USA Center for Manufacturing Innovation, 2Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 3Department of Mechanical Engineering, Boston University, 4Department of Biomedical Engineering, Boston University

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

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Medicine

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice
Richie E. Kohman 1, Xue Han 1, Benjamin S. Bleier 2
1Department of Biomedical Engineering, Boston University, 2Department of Otology and Laryngology, Massachusetts Eye and Ear Infirmary, Harvard Medical School

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

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Medicine

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis
Ajay Kumar *1, Anthony Accorsi *1, Younghwa Rhee 1, Mahasweta Girgenrath 1
1Sargent College, Boston University

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

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Bioengineering

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice
Erika J. Fong 1,2, Chao Huang 1, Julie Hamilton 1, William J. Benett 1, Mihail Bora 1, Alison Burklund 1, Thomas R. Metz 1, Maxim Shusteff 1
1Materials Engineering Division, Lawrence Livermore National Laboratory, 2Department of Biomedical Engineering, Boston University

This protocol describes a system architecture for performing automated small volume (0.15–1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

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JoVE Core

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications
Jonah Kaplan 1, Mark Grinstaff 2
1Department of Biomedical Engineering, Boston University, 2Departments of Biomedical Engineering, Chemistry, and Medicine, Boston University

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

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Bioengineering

Automated Robotic Liquid Handling Assembly of Modular DNA Devices
Luis Ortiz 1,2, Marilene Pavan 2, Lloyd McCarthy 3, Joshua Timmons 3, Douglas M. Densmore 4
1Graduate Program in Molecular Biology, Cell Biology, and Biochemistry, Boston University, 2Biological Design Center, Boston University, 3Lattice Automation, 4Department of Electrical and Computer Engineering, Biological Design Center, Boston University

Here, an automated workflow to perform modular DNA "device" assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

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Engineering

Synthesis of Ionic Liquid Based Electrolytes, Assembly of Li-ion Batteries, and Measurements of Performance at High Temperature
Xinrong Lin 1, Jennifer Chapman Varela 1, Mark W. Grinstaff 1,2,3
1Department of Chemistry, Boston University, 2Department of Biomedical Engineering, Boston University, 3Department of Medicine, Boston University

Here, we describe protocols to prepare phosphonium-based ionic liquid and lithium bis(trifluoromethane)sulfonimide salt electrolytes, and assemble a non-flammable and high temperature functioning lithium-ion coin cell battery.

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Isolation and Detection of Telomeric DNA C-Circles from Mammalian Cells
Emily Mason-Osann 1, Kelli E Cox 1, Rachel Flynn 1
1Department of Pharmacology & Experimental Therapeutics, Boston University

Cancer cells can overcome replicative senescence by activating the alternative lengthening of telomeres (ALT) pathway. ALT positive cancer cells are uniquely characterized by the production of circular C-rich telomeric DNA sequences (C-circle). This protocol describes how to detect C-circles isolated from ALT-positive mammalian cells.

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Behavior

A Within-subjects Experimental Protocol to Assess the Effects of Social Input on Infant EEG
Ashley M. St. John 1, Katie Kao 1, Meia Chita-Tegmark 1, Jacqueline Liederman 1, Philip G. Grieve 2, Amanda R. Tarullo 1
1Department of Psychological and Brain Sciences, Boston University, 2Department of Pediatrics, Columbia University Medical Center

This novel protocol is designed to assess the neural bases of social interaction in infants. The paradigm is designed to tease apart how various social inputs such as language, joint attention, and face-to-face interaction relate to infant neural activation. Infant EEG power is recorded during both social and nonsocial conditions.

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Biology

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum
Anna J. Saltman *1,2, May Barakat *1, Donald M. Bryant 1, Anastasia Brodovskaya 1, Jessica L. Whited 1
1Department of Orthopedic Surgery, Harvard Medical School and Brigham and Women's Hospital, 2Division of Graduate Medical Sciences, Boston University

Using a lipophilic 1,1'-Dioctadecy-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

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Neuroscience

Neurovascular Network Explorer 2.0: A Simple Tool for Exploring and Sharing a Database of Optogenetically-evoked Vasomotion in Mouse Cortex In Vivo
Hana Uhlirova 1,2, Peifang Tian 3,4, Kıvılcım Kılıç 3,5, Martin Thunemann 1, Vishnu B. Sridhar 6, Radim Chmelik 2,7, Hauke Bartsch 1, Anders M. Dale 1,3, Anna Devor 1,3,8, Payam A. Saisan 3
1Department of Radiology, University of California, San Diego, 2Central European Institute of Technology, Brno University of Technology, 3Department of Neurosciences, University of California, San Diego, 4Department of Physics, John Carroll University, 5Department of Biomedical Engineering, Boston University, 6Bioengineering Undergraduate Program, University of California, San Diego, 7Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology, 8Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School

A graphical user interface for exploring and sharing a database of optogenetically-induced vascular responses in mouse somatosensory cortex in vivo measured by 2-photon microscopy is presented. It allows browsing the data, criteria-based selection, averaging, localization of measurements within a 3D volume of vasculature and exporting the data.

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Bioengineering

Preparation, Purification, and Use of Fatty Acid-containing Liposomes
Lin Jin *1,2, Aaron E. Engelhart *1,3, Katarzyna P. Adamala 1,3, Jack W. Szostak 1
1Howard Hughes Medical Institute and Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 2Department of Biomedical Engineering, Boston University, 3Department of Genetics, Cell Biology, and Development, University of Minnesota

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

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Bioengineering

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)
Weiye Song *1, Libo Zhou *1, Ji Yi 1,2
1Department of Medicine, Boston University School of Medicine, 2Department of Biomedical Engineering, Boston University

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

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Bioengineering

Rat Model of Adhesive Capsulitis of the Shoulder
Stephen M. Okajima *1, M. Belen Cubria *1, Sharri J. Mortensen 1, Juan C. Villa-Camacho 1, Philip Hanna 1, Aron Lechtig 1, Miguel Perez-Viloria 1, Patrick Williamson 1, Mark W. Grinstaff *3, Edward K. Rodriguez *2, Ara Nazarian *1,4
1Center for Advanced Orthopaedic Studies, Beth Israel Deaconess Medical Center and Harvard Medical School, 2Carl J. Shapiro Department of Orthopaedic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, 3Departments of Biomedical Engineering, Chemistry, and Medicine, Boston University, 4Department of Orthopaedic Surgery, Yerevan State Medical University

This protocol presents an in vivo rat model of adhesive capsulitis. The model includes an internal fixation of the glenohumeral joint with extra-articular suture fixation for an extended time, resulting in a decreased rotational range of motion (ROM) and increased joint stiffness.

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Genetics

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue
Maria Dafne Cardamone 1, Joseph Orofino 1, Adam Labadorf 2, Valentina Perissi 1
1Biochemistry Department, Boston University School of Medicine, 2Bioinformatic Hub, Boston University

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

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Biochemistry

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale
Melaku Garsamo 1,3, Yun Zhou 2,3, Xing Liu 1,3
1Department of Biochemistry, Purdue University, 2Department of Botany and Plant Pathology, Purdue University, 3Center for Plant Biology, Purdue University

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique.  

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Genetics

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes
Hannah D. Rickner 1, Sheng-Yong Niu 1,2, Christine S. Cheng 1,3
1Department of Biology, Boston University, 2Department of Computer Science and Engineering, University of California San Diego, 3Bioinformatics Program, Boston University

Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer.

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Genetics

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences
Shaleen Shrestha *1, Xing Liu *1, Clarissa Stephanie Santoso *1, Juan Ignacio Fuxman Bass 1,2
1Department of Biology, Boston University, 2Bioinformatics Program, Boston University

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of >1,000 TFs in a single experiment.

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Genetics

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER
Zachary J. Heins 1,2, Christopher P. Mancuso 1,2, Szilvia Kiriakov 1,3, Brandon G. Wong 1,2, Caleb J. Bashor 4, Ahmad S. Khalil 1,2,5
1Biological Design Center, Boston University, 2Department of Biomedical Engineering, Boston University, 3Program in Molecular Biology, Cell Biology and Biochemistry, Boston University, 4Department of Bioengineering, Rice University, 5Wyss Institute for Biologically Inspired Engineering, Harvard University

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

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Chemistry

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases
Brian J. Stockman 1, Abinash Kaur 1, Julia K. Persaud 1, Maham Mahmood 1, Samantha F. Thuilot 1, Melissa B. Emilcar 1, Madison Canestrari 1, Juliana A. Gonzalez 1, Shannon Auletta 1, Vital Sapojnikov 1, Wagma Caravan 1,2, Samantha N. Muellers 1,3
1Department of Chemistry, Adelphi University, 2Department of Chemistry, Washington University in St. Louis, 3Department of Chemistry, Boston University

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 μM and 250 μM, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

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Biology

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior
Ashley L. Comer 1,2,3,4, Balaji Sriram 5, William W. Yen 2, Alberto Cruz-Martín 1,2,3,4,6
1The Graduate Program for Neuroscience, Boston University, 2Department of Biology, Boston University, 3Neurophotonics Center, Boston University, 4Center for Systems Neuroscience, Boston University, 5Research and Early Development, Biogen, 6Department Pharmacology and Experimental Therapeutics, Boston University

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

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Neuroscience

The Impact of Motor Task Conditions on Goal-Directed Arm Reaching Kinematics and Trunk Compensation in Chronic Stroke Survivors
Jaimie Girnis 1,2, Tarek Agag 1, Tobias Nobiling 1,3, Vanessa Sweet 1,4, Bokkyu Kim 1
1Department of Physical Therapy Education, SUNY Upstate Medical University, 2College of Health and Rehabilitation Sciences: Sargent College Center for Neurorehabilitation, Boston University, 3Department of Physical Medicine and Rehabilitation, University of Rochester Medical Center, 4Rehabilitation Today

This protocol is intended to investigate the impact of task conditions on movement strategies in chronic stroke survivors. Further, this protocol can be used to examine if a restriction in elbow extension induced by neuromuscular electrical stimulation causes trunk compensation during goal-directed arm reaches in non-disabled adults.

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Biology

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium
Meng Zhang 1,2, Mohamed N. Seleem 3, Ji-Xin Cheng 1,2,4,5
1Department of Electrical and Computer Engineering, Boston University, 2Boston University Photonics Center, Boston University, 3Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 4Department of Biomedical Engineering, Boston University, 5Department of Chemistry, Boston University

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

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Biology

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry
Amine Belaid 2, Harilaos Filippakis 1
1Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, 2Metcalf Science Center, Chemistry Department, Boston University

This protocol provides experimental tools to evaluate macropinocytic uptake of nutrients (carbohydrate and protein) by mTORC1-hyperactive cells. Detailed steps to quantify the uptake of fluorescently labeled dextran and bovine serum albumin (BSA) are described.

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Bioengineering

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics
Katariina Jaenes *1, Severino Jefferson Ribeiro da Silva *1,2, Justin R. J. Vigar *1, Kaiyue Wu 3,4, Masoud Norouzi 1, Pouriya Bayat 1, Margot Karlikow 1, Seray Cicek 1, Yuxiu Guo 1, Alexander A. Green 3,4, Lindomar Pena 2, Keith Pardee 1,5
1Leslie Dan Faculty of Pharmacy, University of Toronto, 2Laboratory of Virology and Experimental Therapy (LAVITE), Department of Virology, Aggeu Magalhães Institute (IAM), Oswaldo Cruz Foundation (Fiocruz), 3Department of Biomedical Engineering, Boston University, 4Molecular Biology, Cell Biology & Biochemistry Program, Graduate School of Arts and Sciences, Boston University, 5Department of Mechanical and Industrial Engineering, University of Toronto

Access to decentralized, low-cost, and high-capacity diagnostics that can be deployed into the community for decentralized testing is critical for combating global health crises. This manuscript describes how to build paper-based diagnostics for viral RNA sequences that can be detected with a portable optical reader.

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Bioengineering

Pattern Generation for Micropattern Traction Microscopy
Katie A. Bunde 1, Dimitrije Stamenović 1, Michael L. Smith 1
1Department of Biomedical Engineering, Boston University

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

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Immunology and Infection

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis
Linlin Zhang 1,2, Mengjia Liu 1,2, Meng Zhang 1,2, Junhong Ai 1,2, Jiao Tian 1,2, Ran Wang 1,2, Zhengde Xie 1,2
1Beijing Key Laboratory of Pediatric Respiratory Infectious Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, 2Research Unit of Critical Infection in Children, 2019RU016, Chinese Academy of Medical Sciences

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

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Biochemistry

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder
Kristen L. Segars *1, Nicholas A. Azzari *2, Stephanie Gomez 2, Celeste B. Rich 3, Vickery Trinkaus-Randall 2,3
1Department of Pharmacology, School of Medicine, Boston University, 2Department of Biochemistry, School of Medicine, Boston University, 3Department of Ophthalmology, School of Medicine, Boston University

The present work describes a novel experimental protocol that utilizes a 3D printed holder to enable high-resolution live cell imaging of enucleated globes. Through this protocol, the cellular calcium signaling activity in wounded corneal epithelium from ex vivo globes can be observed in real time.

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Immunology and Infection

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique
Linlin Zhang *1,2, Meng Zhang *1,2,3, Mengjia Liu 1,2, Junhong Ai 1,2, Jiao Tian 1,2, Huizheng Ge 4, Ran Wang 1,2, Zhengde Xie 1,2
1Beijing Key Laboratory of Pediatric Respiratory Infectious Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, 2Research Unit of Critical Infection in Children, 2019RU016, Chinese Academy of Medical Sciences, 3Department of Pediatric Rehabilitation, Beijing Boai Hospital, School of Rehabilitation Medicine, Capital Medical University, China Rehabilitation Research Center, 4Center of Excellence (Beijing), Becton Dickinson Medical Devices (Shanghai) Co Ltd

The present protocol combines ex vivo stimulation and flow cytometry to analyze polyfunctional T cell (TPF) profiles in peripheral blood mononuclear cells (PBMCs) within Japanese encephalitis virus (JEV)-vaccinated children. The detection method and flow cytometry color scheme of JEV-specific TPFs were tested to provide a reference for similar studies.

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Cancer Research

Venous Thrombosis Assay in a Mouse Model of Cancer
Saran Lotfollahzadeh 1, Xiaosheng Yang 1, David Jasen Wu Wong 1, Jingyan Han 2, Francesca Seta 2, Suvranu Ganguli 3, Asha Jose 1, Katya Ravid 4,5, Vipul C. Chitalia 1,6,7,8
1Renal Section, Department of Medicine, Chobanian and Avedisian School of Medicine, Boston University, 2Vascular Biology Section, Department of Medicine, Chobanian and Avedisian School of Medicine, Boston University, 3Division of Interventional Radiology, Department of Radiology, Chobanian and Avedisian School of Medicine, Boston University, 4Department of Medicine, Whitaker Cardiovascular Institute, Chobanian and Avedisian School of Medicine, Boston University, 5Department of Biochemistry and Cell Biology, Chobanian and Avedisian School of Medicine, Boston University, 6Veterans Affairs Boston Healthcare System, 7Institute of Medical Engineering and Sciences, Massachusetts Institute of Technology, 8Center of Cross-Organ Vascular Pathology, Department of Medicine, Chobanian and Avedisian School of Medicine, Boston University

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis.

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Bioengineering

A Passive Ankle Dorsiflexion Testing System for an In Vivo Model of Overuse-induced Tendinopathy
Pooja H. Chainani 1,2, Patrick M. Williamson 1,2, Diana Yeritsyan 1, Kaveh Momenzadeh 1, Nadim Kheir 1, Joseph P. DeAngelis 1,3, Arun J. Ramappa 1,3, Ara Nazarian 1,2,3,4
1Musculoskeletal Translational Innovation Initiative, Carl J. Shapiro Department of Orthopaedic Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Mechanical Engineering Department, Boston University, 3Carl J. Shapiro Department of Orthopaedic Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 4Department of Orthopaedic Surgery, Yerevan State Medical University

This protocol presents a testing system used to induce quantifiable and controlled fatigue injuries in a rat Achilles tendon for an in-vivo model of overuse-induced tendinopathy. The procedure consists of securing the rat's ankle to a joint actuator that performs passive ankle dorsiflexion with a custom-written MATLAB script.

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Bioengineering

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics
Audrey E. Van Heest 1, Feiyang Deng 1, Renee T. Zhao 2, Nour Saida Harzallah 2,3, Heather E. Fleming 3,4, Sangeeta N. Bhatia 2,3,4,5,6,7,8, Liangliang Hao 1,2,3
1Department of Biomedical Engineering, Boston University, 2Institute for Medical Engineering and Science, Massachusetts Institute of Technology, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Howard Hughes Medical Institute, 5Broad Institute of Massachusetts Institute of Technology and Harvard, 6Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 7Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 8Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

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Bioengineering

Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)
Zhongyue Guo *1, Yeran Bai *2,3, Fátima C. Pereira 4, Ji-Xin Cheng 1,5
1Department of Biomedical Engineering, Boston University, 2Neuroscience Research Institute, Department of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, 3Photothermal Spectroscopy Corp., 4School of Biological Sciences, University of Southampton, 5Department of Electrical & Computer Engineering, Photonics Center, Boston University

Here, we present a protocol using optical photothermal infrared-fluorescence in situ hybridization (OPTIR-FISH), also known as mid-infrared photothermal-FISH (MIP-FISH), to identify individual cells and understand their metabolism. This methodology can be applied broadly for diverse applications, including mapping cellular metabolism with single-cell resolution.

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Medicine

Profiling Luminal pH in Three-Dimensional Gastrointestinal Organoids Using Microelectrodes
Katrina Lyon 1, Rama Bansil 2, Diane Bimczok 1
1Department of Microbiology & Cell Biology, Montana State University, 2Department of Physics, Boston University

The present protocol describes pH measurements in human tissue-derived gastric organoids using microelectrodes for spatiotemporal characterization of intraluminal physiology.

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