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100 ARTICLES PUBLISHED IN JoVE

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Biology

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging
Tobias D. Henning 1, Sophie Boddington 1, Heike E. Daldrup-Link 1
1Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

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Biology

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging
Sophie Boddington 1, Tobias D. Henning 1, Elizabeth J. Sutton 1, Heike E. Daldrup-Link 1
1Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

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Biology

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells
Kitchener Wilson 1, Jin Yu 1, Andrew Lee 1, Joseph C. Wu 1
1Departments of Radiology and Medicine (Cardiology), Stanford University School of Medicine

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

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Biology

Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes
Austin L Brown 1, Brandon E. Johnson 2, Miriam B. Goodman 2
1Department of Molecular and Cellular Physiology, Stanford University , 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

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Biology

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller
Austin L. Brown 1, Brandon E. Johnson 2, Miriam B. Goodman 2
1Department of Molecular and Cellular Physiology, Stanford University , 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

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Biology

Pressure-polishing Pipettes for Improved Patch-clamp Recording
Brandon E. Johnson 1, Austin L. Brown 1, Miriam B. Goodman 1
1Department of Molecular and Cellular Physiology, Stanford University School of Medicine

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

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Biology

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia
Ngan F. Huang 1, Hiroshi Niiyama 1, Abhijit De 2, Sanjiv S. Gambhir 2, John P. Cooke 1
1Division of Cardiovascular Medicine, Stanford University , 2Department of Radiology, Stanford University

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

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Biology

Murine Model of Hindlimb Ischemia
Hiroshi Niiyama 1, Ngan F. Huang 1, Mark D. Rollins 2, John P. Cooke 1
1Division of Cardiovascular Medicine, Stanford University , 2Department of Anesthesiology, University of California, San Francisco

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

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Biology

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication
Fabian Kloosterman 1,2, Thomas J. Davidson 1,2, Stephen N. Gomperts 1,2, Stuart P. Layton 1,2, Gregory Hale 1,2, David P. Nguyen 1,2, Matthew A. Wilson 1,2
1Picower Institute for Learning and Memory, MIT - Massachusetts Institute of Technology, 2Department of Brain and Cognitive Science, MIT - Massachusetts Institute of Technology

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

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Biology

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly
David P. Nguyen 1,2, Stuart P. Layton 1,2, Gregory Hale 1,2, Stephen N. Gomperts 1,2, Thomas J. Davidson 1,2, Fabian Kloosterman 1,2, Matthew A. Wilson 1,2
1Department of Brain and Cognitive Science, MIT - Massachusetts Institute of Technology, 2Picower Institute for Learning and Memory, MIT - Massachusetts Institute of Technology

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

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Biology

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects
Alexander J. Nedopil 1, Lydia G. Mandrussow 1, Heike E. Daldrup-Link 1
1Department of Radiology and Biomedical Imaging, Medical Center, University of California San Francisco

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

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Medicine

Dissection of Human Vitreous Body Elements for Proteomic Analysis
Jessica M. Skeie 1, Vinit B. Mahajan 1
1Department of Ophthalmology and Visual Sciences, Omics Laboratory, University of Iowa

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

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Biology

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses
Jessica M. Skeie 1,2, Stephen H. Tsang 3, Vinit B. Mahajan 1,2
1Omics Laboratory, University of Iowa, 2Ophthalmology and Visual Sciences, University of Iowa, 3Harkness Eye Institute, Columbia University College of Physicians and Surgeons

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

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Medicine

Mouse Eye Enucleation for Remote High-throughput Phenotyping
Vinit B. Mahajan 1,2, Jessica M. Skeie 1,2, Amir H. Assefnia 2,3, MaryAnn Mahajan 1,2, Stephen H. Tsang 2,4
1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

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Medicine

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle
Rosalinda T. Castaneda 1,2, Aman Khurana 1,2, Ramsha Khan 1,2, Heike E. Daldrup-Link 1
1Department of Radiology, Molecular Imaging Program at Stanford (MIPS) , 2Stanford School of Medicine, Stanford University

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

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Medicine

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate
David D. Lo *1,2, Jeong S. Hyun *1,3, Michael T. Chung 1, Daniel T. Montoro 1, Andrew Zimmermann 1, Monica M. Grova 1,4, Min Lee 5, Derrick C. Wan 1, Michael T. Longaker 1
1Department of Surgery, Stanford University , 2Department of Surgery, Duke University , 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco , 5School of Dentistry, University of California, Los Angeles

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

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Medicine

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye
Katherine J. Wert 1,2, Jessica M. Skeie 3,4, Richard J. Davis 1, Stephen H. Tsang 1,3, Vinit B. Mahajan 3,4
1Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University , 2Institute of Human Nutrition, College of Physicians & Surgeons, Columbia University , 3Omics Laboratory, University of Iowa , 4Department of Ophthalmology and Visual Sciences, University of Iowa

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

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Medicine

Parabiosis in Mice: A Detailed Protocol
Paniz Kamran 1,2, Konstantina-Ioanna Sereti 1,2, Peng Zhao 1,2, Shah R. Ali 3, Irving L. Weissman 3, Reza Ardehali 1,2
1Department of Medicine-Division of Cardiology, University of California, Los Angeles, 2Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

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Bioengineering

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles
Michael Keeney *1, Lorenzo Deveza *1,2, Fan Yang 1,2
1Department of Orthopaedic Surgery, Stanford University , 2Department of Bioengineering, Stanford University

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

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Medicine

Ultrasonic Assessment of Myocardial Microstructure
Pranoti Hiremath 1, Michael Bauer 2, Hui-Wen Cheng 2, Kazumasa Unno 2, Ronglih Liao 2, Susan Cheng 2
1Harvard Medical School, 2Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

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JoVE Journal

Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2
Juan Carlos Polanco 1, Bei Wang 1, Qi Zhou 1, Hun Chy 1, Carmel O'Brien 1, Andrew L. Laslett 1
1Materials Science and Engineering, CSIRO

We describe the use of the monoclonal antibodies TG30 (CD9) and GCTM-2 for the combined detection of cell surface antigens via fluorescence activated cell sorting (FACS) for the identification and enrichment of live human embryonic stem cells (hESC) using positive selection and also the use of negative selection to purge hESCs from a mixed cell population.

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Behavior

Nest Building as an Indicator of Health and Welfare in Laboratory Mice
Brianna N. Gaskill 1, Alicia Z. Karas 2, Joseph P. Garner 3,4, Kathleen R. Pritchett-Corning 1
1Research Models and Services, Charles River, 2Department of Clinical Sciences, Tufts University, 3Department of Comparative Medicine, Stanford University, 4Department of Psychiatry and Behavioral Sciences, Stanford University

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

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Medicine

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography
Hui-Wen Cheng *1,2, Sudeshna Fisch *1, Susan Cheng 1, Michael Bauer 1, Soeun Ngoy 1, Yiling Qiu 1, Jian Guan 1, Shikha Mishra 1, Christopher Mbah 1, Ronglih Liao 1
1Cardiac Muscle Research Labratory, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, 2Cardiovascular Department, Chang Gung Memorial Hospital

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

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Chemistry

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography
Samantha M. Desmarais 1, Felipe Cava 2, Miguel A. de Pedro 3, Kerwyn Casey Huang 1,4
1Department of Bioengineering, Stanford University, 2Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, 3Campus de Cantoblanco, Universidad Autonoma de Madrid, 4Department of Microbiology and Immunology, Stanford University School of Medicine

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

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Neuroscience

In vivo Optogenetic Stimulation of the Rodent Central Nervous System
Michelle M. Sidor 1, Thomas J. Davidson 2, Kay M. Tye 3, Melissa R. Warden 4, Karl Diesseroth 2,5, Colleen A. McClung 1
1Department of Psychiatry, University of Pittsburgh Medical Center, 2Department of Bioengineering, Stanford University, 3Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, 4Department of Neurobiology and Behavior, Cornell University, 5Department of Psychiatry and Behavioral Sciences, Stanford University

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

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Biology

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions
Joseph Fasolo 1, Hogune Im 1, Michael P. Snyder 1,2
1Department of Genetics, Stanford University, 2Stanford Center for Genomics and Personalized Medicine, Stanford University

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

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Neuroscience

Paired Whole Cell Recordings in Organotypic Hippocampal Slices
Chantelle Fourie 1, Marianna Kiraly 2, Daniel V. Madison *2, Johanna M. Montgomery *1
1Department of Physiology and Centre for Brain Research, University of Auckland, 2Department of Molecular and Cellular Physiology, Stanford University

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

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Biology

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Subarna Bhattacharya *1, Paul W. Burridge *2, Erin M. Kropp 1, Sandra L. Chuppa 1, Wai-Meng Kwok 3, Joseph C. Wu 2, Kenneth R. Boheler 4,5, Rebekah L. Gundry 1,6
1Department of Biochemistry, Medical College of Wisconsin, 2Stanford Cardiovascular Institute, Stanford University School of Medicine, 3Department of Anesthesiology, Medical College of Wisconsin, 4Stem Cell and Regenerative Medicine Consortium, LKS Faculty of Medicine, Hong Kong University, 5Division of Cardiology, Johns Hopkins University School of Medicine, 6Cardiovascular Research Center, Biotechnology and Bioengineering Center, Medical College of Wisconsin

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

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JoVE Journal

Osteoclast Derivation from Mouse Bone Marrow
Ruth Tevlin *1, Adrian McArdle *1,2, Charles K.F. Chan 2, John Pluvinage 2, Graham G. Walmsley 1,2, Taylor Wearda 1,2, Owen Marecic 1,2, Michael S. Hu 1, Kevin J. Paik 1, Kshemendra Senarath-Yapa 1, David A. Atashroo 1, Elizabeth R. Zielins 1, Derrick C. Wan 1, Irving L. Weissman 1,2, Michael T. Longaker 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

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Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis
Elizabeth R. Zielins *1, Ruth Tevlin *1, Michael S. Hu 1, Michael T. Chung 1, Adrian McArdle 1, Kevin J. Paik 1, David Atashroo 1, Christopher R. Duldulao 1, Anna Luan 1, Kshemendra Senarath-Yapa 1, Graham G. Walmsley 1, Taylor Wearda 1, Michael T. Longaker 1,2, Derrick C. Wan 1
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

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Medicine

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography
David A. Atashroo *1, Kevin J. Paik *1, Michael T. Chung 1, Adrian McArdle 1, Kshemendra Senarath-Yapa 1, Elizabeth R. Zielins 1, Ruth Tevlin 1, Christopher R. Duldulao 1, Graham G. Walmsley 1, Taylor Wearda 1, Owen Marecic 1, Michael T. Longaker 1,2, Derrick C. Wan 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

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Medicine

A Mouse Fetal Skin Model of Scarless Wound Repair
Graham G. Walmsley *1,2, Michael S. Hu *1,2,3, Wan Xing Hong 1,4, Zeshaan N. Maan 1, H. Peter Lorenz 1, Michael T. Longaker 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Surgery, John A. Burns School of Medicine, University of Hawai'i, 4University of Central Florida College of Medicine

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

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Medicine

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats
Sudeshna Fisch 1, Ronglih Liao 1, Li-Li Hsiao 2, Tzongshi Lu 2
1Cardiac Muscle Research Laboratory, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 2Renal Division, Brigham and Women's Hospital, Harvard Medical School

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

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Behavior

Infant Auditory Processing and Event-related Brain Oscillations
Gabriella Musacchia 1,2,3, Silvia Ortiz-Mantilla 1, Teresa Realpe-Bonilla 1, Cynthia P. Roesler 1, April A. Benasich 1
1Center for Molecular & Behavioral Neuroscience, Rutgers University, State University of New Jersey, Newark, 2Department of Audiology, University of the Pacific, 3Department of Otolaryngology, Head & Neck Surgery, Stanford University

High-density electroencephalography (dEEG) is being used increasingly to study brain development and plasticity in the early years of life. Here we present an application of sophisticated analysis techniques that builds on traditional EEG recording to understand the oscillatory dynamics of rapid auditory processing in the infant brain.

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Medicine

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice
Wei-Ting Chang *1,2, Sudeshna Fisch *1, Michael Chen 1, Yiling Qiu 1, Susan Cheng 1, Ronglih Liao 1
1Cardiac Muscle Research Laboratory, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 2Division of Cardiovascular Medicine, Chi-Mei Medical Center, Tainan

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

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Developmental Biology

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation
Arun Sharma *1, Guang Li *1, Kuppusamy Rajarajan *1, Ryoko Hamaguchi 1, Paul W. Burridge 1, Sean M. Wu 1,2
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Institute of Stem Cell Biology and Regenerative Medicine, Cardiovascular Medicine Division, Department of Medicine, Child Health Research Institute, Stanford University School of Medicine

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

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Immunology and Infection

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice
Nicolas Gaudenzio 1, Riccardo Sibilano 1, Philipp Starkl 1, Mindy Tsai 1, Stephen J. Galli 1,2, Laurent L. Reber 1
1Department of Pathology, Stanford University School of Medicine, 2Department of Microbiology & Immunology, Stanford University School of Medicine

We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

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Medicine

Reduction of Iatrogenic Atrial Septal Defects with an Anterior and Inferior Transseptal Puncture Site when Operating the Cryoballoon Ablation Catheter
Michael E. Rich 1, Andrew Tseng 2, Hae W. Lim 3, Paul J. Wang 4, Wilber W. Su 1
1Department of Cardiovascular Medicine, Cavanagh Heart Center, Banner-University Medical Center, 2Mayo Medical School, Mayo Clinic, 3AF Solutions, Medtronic plc, 4Cardiology Division, Stanford University School of Medicine, Stanford University

The goal of this study is to demonstrate the preferential location of transseptal puncture during a cryoballoon catheter ablation procedure for the treatment of atrial fibrillation.

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Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains
Thomas Gaj *1, Jia Liu *1,2
1Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute, 2Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

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JoVE Core

Fabrication and Operation of a Nano-Optical Conveyor Belt
Jason Ryan 1, Yuxin Zheng 1, Paul Hansen 1, Lambertus Hesselink 2
1Electrical Engineering, Stanford University, 2Applied Physics, Stanford University

The scalability and resolution of conventional optical manipulation techniques are limited by diffraction. We circumvent the diffraction limit and describe a method of optically transporting nanoparticles across a chip using a gold surface patterned with a path of closely spaced C-shaped plasmonic resonators.

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Bioengineering

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation
Piera Smeriglio *1, Janice H. Lai *1,2, Fan Yang 1,3, Nidhi Bhutani 1
1Orthopaedic Surgery Department, Stanford University, 2Mechanical Engineering Department, Stanford University, 3Bioengineering Department, Stanford University

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

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Neuroscience

Optogenetic Functional MRI
Peter Lin 1, Zhongnan Fang 2, Jia Liu 1, Jin Hyung Lee 1,2
1Neurology and Neurological Sciences, Stanford University, 2Electrical Engineering, Neurology and Neurological Sciences, Stanford University

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

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Developmental Biology

Murine Dermal Fibroblast Isolation by FACS
Graham G. Walmsley *1,2, Zeshaan N. Maan *1, Michael S. Hu *1,2,3, David A. Atashroo 1, Alexander J. Whittam 1, Dominik Duscher 1, Ruth Tevlin 1, Owen Marecic 1, H. Peter Lorenz 1, Geoffrey C. Gurtner 1, Michael T. Longaker 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Surgery, John A. Burns School of Medicine, University of Hawai'i

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

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Developmental Biology

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery
Martin H. M. Sailer 1, Durga Sarvepalli 2, Catherine Brégère 3, Urs Fisch 3, Marin Guentchev 4, Michael Weller 6, Raphael Guzman 3, Bernhard Bettler 1, Arkasubhra Ghosh *2, Gregor Hutter *5
1Dept. of Biomedicine, Pharmacenter, University of Basel, 2Molecular Signalling and Gene Therapy, Narayana Nethralaya Foundation, Narayana Health City, 3Brain Ischemia and Regeneration, Department of Biomedicine, University Hospital Basel, 4Department of Neurosurgery, Klinikum Idar-Oberstein, 5Department of Neurosurgery and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 6Department of Neurology, Laboratory of Molecular Neuro Oncology, University Hospital of Zurich

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

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Medicine

Creation of Abdominal Adhesions in Mice
Clement D. Marshall 1, Michael S. Hu 1, Tripp Leavitt 1, Leandra A. Barnes 1, Alexander T.M. Cheung 1, Samir Malhotra 1, H. Peter Lorenz 1, Michael T. Longaker 1
1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

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Biology

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse
Ke Yuan 1,2, Mark E. Orcholski 1,2, Ngan F. Huang 2,3, Vinicio A. de Jesus Perez 1,2
1Division of Pulmonary and Critical Care Medicine, School of Medicine, Stanford University, 2Stanford Cardiovascular Institute, School of Medicine, Stanford University, 3VA Palo Alto Health Care System, Department of Cardiothoracic Surgery, School of Medicine, Stanford University

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

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Bioengineering

Quantification of Strain in a Porcine Model of Skin Expansion Using Multi-View Stereo and Isogeometric Kinematics
Adrian Buganza Tepole 1, Elbert E. Vaca 2, Chad A. Purnell 2, Michael Gart 2, Jennifer McGrath 2, Ellen Kuhl 3, Arun K. Gosain 2
1Mechanical Engineering, Purdue University, 2Division of Plastic Surgery, Ann and Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, 3Mechanical Engineering, Bioengineering, Cardiothoracic Surgery, Stanford University

This protocol uses multi-view stereo to generate three-dimensional (3D) models out of uncalibrated sequences of photographs, making it affordable and adjustable to a surgical setting. Strain maps between the 3D models are quantified with spline-based isogeometric kinematics, which facilitate representation of smooth surfaces over coarse meshes sharing the same parameterization.

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Developmental Biology

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model
Clement D. Marshall 1, Elizabeth R. Zielins 1, Elizabeth A. Brett 1, Charles P. Blackshear 1, Michael S. Hu 1, Tripp Leavitt 1, Leandra A. Barnes 1, H. Peter Lorenz 1, Michael T. Longaker 1, Derrick C. Wan 1
1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

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Bioengineering

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices
Kara Brower *1,2,4, Adam K. White *1,2, Polly M. Fordyce 1,2,3,4
1Department of Bioengineering, Stanford University, 2Microfluidic Foundry, Stanford University, 3Department of Genetics, Stanford University, 4Chem-H Institute, Stanford University

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

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Description of a Novel, Surgically Implanted Neuromodulatory Technique Known As Bilateral Epidural Prefrontal Cortical Stimulation (Epcs) for Treatment-Resistant Depression (TRD)
Nolan R. Williams 4, Jaspreet Pannu *4, Brandon S. Bentzley *4, Thomas Hopkins *1, Bashar W. Badran 1, E. Baron Short 1, Mark S. George 1,2,3, Istvan Takacs 2, Ziad Nahas 5
1Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, 2Department of Neurosciences, Medical University of South Carolina, 3Ralph H. Johnson VA Medical Center, 4Department of Psychiatry and Behavioral Sciences, Stanford University, 5American University of Beirut Medical Center

We describe the implantation of 4 epidural stimulation paddles directly above the dura mater over both the left and right frontopolar and dorsolateral prefrontal cortices. Placement was verified using postoperative computed tomography (CT) coregistered with presurgical magnetic resonance imaging (MRI).

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Biology

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary
Wei Hu *1, Amin Tamadon *1, Aaron J.W. Hsueh 2, Yi Feng 1
1Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences; Institutes of Brain Science, Brain Science Collaborative Innovation Center, State Key Laboratory of Medical Neurobiology, Institute of Acupuncture and Moxibustion, Fudan Institutes of Integrative Medicine, Fudan University, 2Program of Reproductive and Stem Cell Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

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Immunology and Infection

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4
Sharon A. Sagan *1,2, Andrés Cruz-Herranz *1, Collin M. Spencer 1,2, Peggy P. Ho 3, Lawrence Steinman 3, Ari J. Green 1, Raymond A. Sobel 4, Scott S. Zamvil 1,2
1Department of Neurology, University of California, 2Program in Immunology, University of California, 3Department of Neurology and Neurological Sciences, Stanford University, 4Department of Pathology, Stanford University

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

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Biochemistry

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis
Thiago Cabral *1,2,7,8, Marcus A. Toral *3,4, Gabriel Velez 3,4, James E. DiCarlo 1,2, Anuradha M. Gore 3, MaryAnn Mahajan 3, Stephen H. Tsang 1,2, Alexander G. Bassuk 5,6, Vinit B. Mahajan 3,9
1Barbara & Donald Jonas Stem Cell Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, 2Edward S. Harkness Eye Institute, New York-Presbyterian Hospital, 3Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University, 4Medical Scientist Training Program, University of Iowa, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7Department of Ophthalmology, Federal University of Sao Paulo (UNIFESP), 8Department of Ophthalmology, Federal University of EspÍrito Santo (UFES), 9Palo Alto Veterans Administration, Palo Alto, CA

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

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Neuroscience

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans
Holger Fehlauer *1, Adam L. Nekimken *1,2, Anna A. Kim 1,2, Beth L. Pruitt 1,2,3, Miriam B. Goodman 1,2, Michael Krieg 4
1Department of Molecular and Cellular Physiology, Stanford University, 2Department of Mechanical Engineering, Stanford University, 3Department of Bioengineering, Stanford University, 4Group of Neurophotonics and Mechanical Systems Biology, The Institute of Photonic Sciences (ICFO)

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

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Neuroscience

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium
Hannah Y. Collins 1, Christopher J. Bohlen 1
1Department of Neurobiology, Stanford University

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

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Cancer Research

Whole-body PET/MRI of Pediatric Patients: The Details That Matter
Anuj Pareek 1, Anne M. Muehe 1, Ashok J. Theruvath 1, Praveen K. Gulaka 1, Sheri L. Spunt 2, Heike E. Daldrup-Link 1
1Department of Radiology, Pediatric Molecular Imaging Program at Stanford (PEDS-MIPS), Stanford University, 2Department of Pediatrics, Stanford University

This article provides comprehensive step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI scans for cancer staging of pediatric patients. The protocol was developed for children above 6 years, or old enough to comply with breath-hold instructions, but can be used for general anesthesia patients as well.

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Cancer Research

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes
Nadine Santana-Magal *1, Diana Rasoulouniriana *1, Corey Saperia 1, Amit Gutwillig 1, Peleg Rider 1, Edgar G. Engleman 2, Yaron Carmi 1
1Department of Pathology, Sackler School of Medicine, Tel-Aviv University, 2Department of Pathology, School of Medicine, Stanford University

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

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Medicine

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke
Aisling M. Chaney 1, Emily M. Johnson 1, Haley C. Cropper 1, Michelle L. James 1,2
1Department of Radiology, Stanford University, 2Department of Neurology and Neurological Sciences, Stanford University

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

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Chemistry

Synthesis and Performance Characterizations of Transition Metal Single Atom Catalyst for Electrochemical CO2 Reduction
Kun Jiang 1, Guangxu Chen 2, Haotian Wang 1
1Rowland Institute, Harvard University, 2Materials Science and Engineering, Stanford University

Here, we present a protocol for the synthesis and electrochemical testing of transition metal single atoms coordinated in graphene vacancies as active centers for selective carbon dioxide reduction to carbon monoxide in aqueous solutions.

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Medicine

Radiation Planning Assistant - A Streamlined, Fully Automated Radiotherapy Treatment Planning System
Laurence E. Court 1, Kelly Kisling 1, Rachel McCarroll 1, Lifei Zhang 1, Jinzhong Yang 1, Hannah Simonds 2, Monique du Toit 2, Chris Trauernicht 2, Hester Burger 3, Jeannette Parkes 3, Mike Mejia 4, Maureen Bojador 4, Peter Balter 1, Daniela Branco 1, Angela Steinmann 1, Garrett Baltz 1, Skylar Gay 1, Brian Anderson 1, Carlos Cardenas 1, Anuja Jhingran 5, Simona Shaitelman 5, Oliver Bogler 6, Kathleen Schmeller 7, David Followill 1, Rebecca Howell 1, Christopher Nelson 1, Christine Peterson 8, Beth Beadle 5,9
1Department of Radiation Physics, University of Texas MD Anderson Cancer Center, 2Department of Radiation Oncology, Stellenbosch University and Tygerberg Hospital, 3Departments of Radiation Oncology and Medical Physics, Groote Schuur Hospital and University of Cape Town, 4Department of Radiation Oncology, University of Santo Tomas Hospital, Benavides Cancer Institute, 5Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, 6Academic Affairs, University of Texas MD Anderson Cancer Center, 7Department of Gynecological Oncology and Reproductive Medicine, University of Texas MD Anderson Cancer Center, 8Department of Biostatistics, University of Texas MD Anderson Cancer Center, 9Department of Radiation Oncology, Stanford University

Radiation therapy is a highly complex cancer treatment that requires multiple specialists to create a treatment plan and provide quality assurance (QA) prior to delivery to a patient. This protocol describes the use of a fully automated system, the Radiation Planning Assistant (RPA), to create high-quality radiation treatment plans.

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Chemistry

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering
Jinpeng Wu 1,2, Shawn Sallis 2,3, Ruimin Qiao 2, Qinghao Li 2,4, Zengqing Zhuo 2,5, Kehua Dai 2,6, Zixuan Guo 2,7, Wanli Yang 2
1Geballe Laboratory for Advanced Materials, Stanford University, 2Advanced Light Source, Lawrence Berkeley National Laboratory, 3Department of Materials Science and Engineering, Binghamton University, 4School of Physics, National Key Laboratory of Crystal Materials, Shandong University, 5School of Advanced Materials, Peking University Shenzhen Graduate School, 6School of Metallurgy, Northeastern University, 7Department of Chemical Engineering, University of California-Santa Barbara

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

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Bioengineering

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D
Bauer L. LeSavage *1, Nicholas A. Suhar *2, Christopher M. Madl 1, Sarah C. Heilshorn 2
1Department of Bioengineering, Stanford University, 2Department of Materials Science and Engineering, Stanford University

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

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Engineering

A Pipette-Tip Based Method for Seeding Cells to Droplet Microfluidic Platforms
Nidhi Sinha *1, Nikita Subedi *1, Florian Wimmers *2, Melf Soennichsen 1, Jurjen Tel 1
1Department of Biomedical Engineering and Institute for Complex Molecular Systems, Laboratory of Immunoengineering, Eindhoven University of Technology, 2Institute for Immunity, Transplantation, and Infection, Beckman Center, Stanford University

This article presents a protocol for seeding scarce population of cells using pipette-tips to droplet microfluidic devices in order to provide higher encapsulation efficiency of cells in droplets.

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JoVE Journal

Imaging FITC-dextran as a Reporter for Regulated Exocytosis
Ofir Klein 1, Amit Roded 1, Koret Hirschberg 2, Mitsunori Fukuda 3, Stephen J. Galli 4, Ronit Sagi-Eisenberg 1
1Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, 2Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, 3Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, 4Departments of Pathology and of Microbiology and Immunology and Sean N. Parker Center for Allergy and Asthma Research, School of Medicine, Stanford University

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

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Medicine

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension
David Boulate 1, Myriam Amsallem 1,2, Jean-Baptiste Menager 1, Simon Dang Van 1, Peter Dorfmuller 3, Andrew Connolly 4, Alban Todesco 5, Benoit Decante 1, Elie Fadel 1, Francois Haddad 2, Olaf Mercier 1
1Research and Innovation Unit, RHU BioArt Lung 2020, Marie Lannelongue Hospital, 2Phenotypic and Biomarker Core Laboratory, Cardiovascular Institute, Stanford University, 3Department of pathology, Marie Lannelongue Hospital, 4Department of pathology, UCSF School of Medicine, 5Department of thoracic surgery, Hopital Nord, APHM, Aix-Marseille University

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics or to improve the understanding of acute right heart failure pathophysiology.

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Immunology and Infection

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning
Jonghee Yoon 1, YoungJu Jo 2,3,4,7, Young Seo Kim 3,4,5, Yeongjin Yu 2,3, Jiyeon Park 6, Sumin Lee 4, Wei Sun Park 2,3, YongKeun Park 2,3,4
1Department of Physics, University of Cambridge, 2Department of Physics, Korea Advanced Institute of Science and Technology, 3KAIST Institute for Health Science and Technology, Korea Advanced Institute of Science and Technology, 4Tomocube, Inc., 5Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 6Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 7Department of Applied Physics, Stanford University

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

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Bioengineering

High-resolution Patterned Biofilm Deposition Using pDawn-Ag43
Xiaofan Jin 1, Ingmar H. Riedel-Kruse 1
1Department of Bioengineering, Stanford University

We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct.

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Cancer Research

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels
Steven M Alves 1, Tian Zhu 1, Anastasia Shostak 1, Ninna S. Rossen 2, Marjan Rafat 1,3,4
1Department of Chemical and Biomolecular Engineering, Vanderbilt University, 2Department of Radiation Oncology, Stanford University, 3Depattment of Biomedical Engineering, Vanderbilt University, 4Department of Radiation Oncology, Vanderbilt University Medical Center

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

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Cancer Research

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research
Jennifer C. Wischhusen 1, Katheryne E. Wilson 2
1Apoptosis, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Centre Léon Bérard, 2Department of Radiology/Molecular Imaging Program at Stanford, School of Medicine, Stanford University

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

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Medicine

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation
Junjie Liang *1,2, Yuanyuan Guo *1,2, Yuxin Zheng 1,2, Shijuan Lang 1, Hongxin Chen 1,2, Yaoyao You 1,2, Bryan O’Young 3,4, Haining Ou *1,2, Qiang Lin *1,2
1Department of Rehabilitation, The Fifth Affiliated Hospital of Guangzhou Medical University, 2Experiment Education Model Center of Rehabilitation Medicine, Guangzhou Medical University, 3Department of Physical Medicine and Rehabilitation, Geisinger Health System, 4Department of Rehabilitation Medicine, New York University School of Medicine

Here, based on a clinician’s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

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Genetics

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis
Carly V. Weiss 1,2, Julie N. Chuong 3, Rachel B. Brem 1,3
1Department of Plant and Microbial Biology, University of California Berkeley, 2Department of Biology, Stanford University, 3Buck Institute for Research on Aging

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

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Environment

Laser-Induced Fluorescence Emission (L.I.F.E.) as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats
Klemens Weisleitner 1,2, Lars Hunger 3, Christoph Kohstall 4, Albert Frisch 5, Michael C. Storrie-Lombardi 6, Birgit Sattler 1,2
1Institute of Ecology, University of Innsbruck, 2Austrian Polar Research Institute, University of Vienna, 3BrainLinks-BrainTools, Bernstein Center Freiburg, 4Atom Science, Kasevich Lab, Stanford University, 5Institute of Experimental Physics, University of Innsbruck, 6Department of Physics, Extraterrestrial Vehicle Instruments Laboratory, Harvey Mudd College

Carbon fluxes in the cryosphere are hardly assessed yet but are crucial regarding climate change. Here we show a novel prototype device that captures the phototrophic potential in supraglacial environments based on laser-induced fluorescence emission (L.I.F.E.) technology offering high spectral and spatial resolution data under in situ conditions.

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Medicine

In Vitro Model of Coronary Angiogenesis
Colton L. Large 1, Halie E. Vitali 1, Jeffery D. Whatley 1, Kristy Red-Horse 2, Bikram Sharma 1
1Department of Biology, Ball State University, 2Department of Biology, Stanford University

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

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Immunology and Infection

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection
Christiaan R. de Vries *1, Johanna M. Sweere *1,2, Heather Ishak 1,3, Vivekananda Sunkari 1, Michelle S. Bach 1, Dan Liu 1, Robert Manasherob 1, Paul L. Bollyky 1,2
1Division of Infectious Diseases, School of Medicine, Stanford University, 2Stanford Immunology, Stanford University, 3Palo Alto Veterans Institute of Research

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

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Developmental Biology

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils
Soah Lee *1,2,3, Huaxiao Yang *1,2,3, Caressa Chen *1,2,3, Sneha Venkatraman 1,2,3, Adrija Darsha 1,2,3, Sean M. Wu 1,2,3, Joseph C. Wu 1,2,3, Timon Seeger 1,2,3,4,5
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiovascular Medicine, Stanford University, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 4Department of Medicine III, University Hospital Heidelberg, 5German Centre for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

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Developmental Biology

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function
Alessandro Evangelisti 1, Katharina Schimmel 1, Shaurya Joshi 2, Kavya Shah 1, Sudeshna Fisch 2, Kevin M. Alexander 1, Ronglih Liao 1, Isabel Morgado 1
1Stanford Cardiovascular Institute, Stanford University, 2Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

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Neuroscience

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry
Wilson Wang *1, Yanrong Zhang *2, Matthew J. Anzivino 1, Edward H. Bertram 3, James Woznak 1,4, Alexander Klibanov 5, Erik Dumont 6, Max Wintermark *2, Kevin S. Lee *1,7,8
1Department of Neuroscience, University of Virginia, 2Department of Radiology, Stanford University, 3Department of Neurology, University of Virginia, 4Global Internship Program, Focused Ultrasound Foundation, 5Department of Medicine, University of Virginia, 6Image Guided Therapy, 7Department of Neurosurgery, University of Virginia, 8Center for Brain, Immunology, and Glia, University of Virginia

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

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Neuroscience

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice
Maja Djurisic 1
1Department of Biology, Neurobiology, and Bio-X, Stanford University

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

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Immunology and Infection

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells
Xiaoyue Sun *1,2,3, Xiaoqiang Yang *1,2,3,4, Chui Zeng 1,2,3, Fayed Attia Koutb Megahed 1,2,3,5, Qi Zhou 1,2,3, Tingdang Liu 1,2,3, Waqas Iqbal 1,2,3, Pingnan Sun 1,2,3, Xiaoling Zhou 1,2,3
1Stem Cell Research Center, Shantou University Medical College, 2The Center for Reproductive Medicine, Shantou University Medical College, 3Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, 4Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 5Department of Nucleic Acid Researches, Genetic Engineering and Biotechnology Research Institute, General Autority - City of Scientific Researches and Technological Applications

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4α, RXRα and PPARα) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

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Chemistry

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril
Weng-I Katherine Chio 1, Gemma Davison 1, Tabitha Jones 1, Jia Liu 1, Ivan P. Parkin 1, Tung-Chun Lee 1,2
1Department of Chemistry, University College London (UCL), 2Institute for Materials Discovery, University College London (UCL)

A host-guest complex of cucurbit[7]uril and uric acid was formed in an aqueous solution before adding a small amount into Au NP solution for quantitative surface-enhanced Raman spectroscopy (SERS) sensing using a modular spectrometer.

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Bioengineering

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells
Dilip Thomas 1, Hyeonyu Kim 1, Nicole Lopez 1, Joseph C. Wu 1,2,3
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, 3Department of Radiology, Stanford University School of Medicine

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

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Developmental Biology

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities
Qixiao Jiang 1, Xiaohui Xu 1, Jamie C. DeWitt 2, Yuxin Zheng 1
1School of Public Health, Qingdao University, 2Department of Pharmacology & Toxicology, Brody School of Medicine, East Carolina University

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

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Bioengineering

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications
Catherine M. Meis *1, Abigail K. Grosskopf *2, Santiago Correa 1, Eric A. Appel 1,3,4
1Department of Materials Science & Engineering, Stanford University, 2Department of Chemical Engineering, Stanford University, 3Department of Bioengineering, Stanford University, 4Department of Pediatrics - Endocrinology, Stanford University

This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated.

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Biochemistry

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography
Claudine Bisson 1,2, Corey W. Hecksel 3,4, James B. Gilchrist 3, M. Alejandra Carbajal 1, Roland A. Fleck 1
1Centre for Ultrastructural Imaging, New Hunt’s House, Guy’s Campus, King’s College London, 2Department of Biological Science, Birkbeck College, University of London, 3Electron Bio-Imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus, 4SLAC National Accelerator Laboratory, Stanford University

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

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Medicine

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies
Timothy Klouda 1, Hyunbum Kim 1, Jiwon Kim 1, Gary Visner 1, Ke Yuan 1
1Divisions of Pulmonary Medicine, Boston Children's Hospital

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

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Immunology and Infection

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells
Qi Zhou *1,2,3, Xiaoling Xie *1,2,3, Zhiqian Zhong 1,2,3, Pingnan Sun 1,2,3, Xiaoling Zhou 1,2,3
1Stem Cell Research Center, Shantou University Medical College, 2The Center for Reproductive Medicine, Shantou University Medical College, 3Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

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Developmental Biology

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila
Tongchao Li 1, Liqun Luo 1
1Department of Biology, Howard Hughes Medical Institute, Department of Biology, Stanford University

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

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Neuroscience

In Vivo Wireless Optogenetic Control of Skilled Motor Behavior
Diana L. Rodriguez-Munoz 1, Omar Jaidar 2, Marcela Palomero-Rivero 1, Mario A. Arias-Garcia 3, Gordon W. Arbuthnott 4, Violeta G. Lopez-Huerta 1
1Institute of Cellular Physiology, National University of Mexico, 2Department of Neurosurgery, Stanford University, 3Facultad de Psicologia, National University of Mexico, 4Brain Mechanisms for Behaviour Unit, Okinawa Institute of Science and Technology Graduate University

The present protocol describes how to use wireless optogenetics combined with high-speed videography in a single pellet reach-to-grasp task to characterize the neural circuits involved in the performance of skilled motor behavior in freely moving mice.

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Medicine

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis
Weiguo Fan 1, Yuan Li 1, Koshi Kunimoto 1, Natalie J. Török 1
1Gastroenterology and Hepatology, Stanford University

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

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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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Bioengineering

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue
Mohammad Mir 1, Jiawen Chen 1, Meghan R. Pinezich 1, John D. O’Neill 3, Brandon A. Guenthart 4, Gordana Vunjak-Novakovic 2, Jinho Kim 1
1Department of Biomedical Engineering, Stevens Institute of Technology, 2Department of Biomedical Engineering, Columbia University, 3Department of Cell Biology, State University of New York Downstate Medical Center, 4Department of Cardiothoracic Surgery, Stanford University

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

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Neuroscience

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography
Samantha T. Reyes *1, E. Carmen Azevedo *1, Haley C. Cropper 1, Sydney Nagy 1, Emily M. Deal 1, Aisling M. Chaney 1, Michelle L. James 1,2
1Department of Radiology, Stanford University, 2Department of Neurology and Neurological Sciences, Stanford University

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

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Bioengineering

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs
Meagan J. Makarcyzk 1,2, Zhong Alan Li 1,3, Ilhan Yu 1, Haruyo Yagi 1, Xiurui Zhang 1, Lauren Yocum 1, Eileen Li 1, Madalyn R. Fritch 1, Qi Gao 4, Bruce A. Bunnell 5, Stuart B. Goodman 4,6, Rocky S. Tuan 1,8, Peter G. Alexander 1,7, Hang Lin 1,2,7
1Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 2Department of Bioengineering, University of Pittsburgh Swanson School of Engineering, 3Department of Neurobiology, University of Pittsburgh School of Medicine, 4Department of Orthopaedic Surgery, Stanford University, 5Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, 6Department of Bioengineering, Stanford University, 7McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, 8The Chinese University of Hong Kong

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

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Medicine

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Shane Rui Zhao 1,2, Gema Mondéjar-Parreño 1,2, Dong Li 1,2, Mengcheng Shen 1,2, Joseph C. Wu 1,2,3
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, 3Department of Radiology, Stanford University School of Medicine

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

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Engineering

Triplet Fusion Upconversion Nanocapsule Synthesis
Tracy H. Schloemer 1,2, Samuel N. Sanders 1, Qi Zhou 2, Pournima Narayanan 3, Manchen Hu 2, Mahesh K. Gangishetty 1, Daniel Anderson 1, Michael Seitz 1,2, Arynn O. Gallegos 2, R. Christopher Stokes 1, Daniel N. Congreve 1,2
1Rowland Institute, Harvard University, 2Department of Electrical Engineering, Stanford University, 3Department of Chemistry, Stanford University

This protocol details the synthesis of upconversion nanocapsules for subsequent use in photopolymerizable resins for triplet fusion upconversion-facilitated volumetric 3D printing.

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Medicine

Radiation Planning Assistant - A Web-based Tool to Support High-quality Radiotherapy in Clinics with Limited Resources
Laurence Edward Court 1, Ajay Aggarwal 2, Hester Burger 3, Carlos Cardenas 4, Christine Chung 1, Raphael Douglas 1, Monique du Toit 5, Anuja Jhingran 1, Raymond Mumme 1, Sikudhani Muya 6, Komeela Naidoo 5, Jerry Ndumbalo 6, Tucker Netherton 1, Callistus Nguyen 1, Adenike Olanrewaju 1, Jeannette Parkes 3, Willie Shaw 7, Christoph Trauernicht 5, Melody Xu 8, Jinzhong Yang 1, Lifei Zhang 1, Hannah Simonds 9, Beth M. Beadle 10
1The University of Texas MD Anderson Cancer Center, 2Guy’s and St. Thomas’ Hospital, 3Groote Schuur Hospital and University of Cape Town, 4University of Alabama at Birmingham, 5Tygerberg Hospital and Stellenbosch University, 6Ocean Road Cancer Institute, 7University of the Free State, 8University of California-San Francisco, 9University Hospitals Plymouth NHS Trust, 10Stanford University

This protocol describes a series of automated tools designed for high-quality radiotherapy autocontouring and autoplanning that are being packaged into a web-based service to maximize robustness and scalability while minimizing operational costs.

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Biology

Rapid Setup of Tissue Microarray and Tiled Area Imaging on the Multiplexed Ion Beam Imaging Microscope Using the Tile/SED/Array Interface
Hadeesha Piyadasa *1, Benjamin Oberlton *1,2, Alex Kong *1, Christine Camacho Fullaway 1, Sricharan Reddy Varra 1, Cameron Sowers 1, Albert G Tsai 1
1Department of Pathology, Stanford University, 2Immunology Graduate Program, Stanford University

Multiplexed ion beam imaging (MIBI) is often used to image tissue microarrays and tiled, contiguous tissue areas, but current software for setting up these experiments is cumbersome. The tile/SED/array interface is an intuitive, interactive graphical tool developed to dramatically simplify and accelerate MIBI run setup.

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Medicine

Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses
Julian Wolf 1,2, Teja Chemudupati 1,2, Aarushi Kumar 1,2, Ditte K. Rasmussen 1,2, Karen M. Wai 2, Robert T. Chang 2, Artis A. Montague 2, Peter H. Tang 3,4, Alexander G. Bassuk 5,6,7, Antoine Dufour 8,9, Prithvi Mruthrunjaya 2, Vinit B. Mahajan 1,2,10
1Molecular Surgery Laboratory, Stanford University, 2Department of Ophthalmology, Byers Eye Institute, Stanford University, 3Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 4Retina Consultants of Minnesota, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7The Iowa Neuroscience Institute (INI), University of Iowa, 8Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, 9Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 10Veterans Affairs Palo Alto Health Care System

This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics.

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Cancer Research

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing
Deshka S. Foster 1, Michelle Griffin 1, Michael Januszyk 1, Daniel Delitto 1, Jeffrey A. Norton 1, Michael T. Longaker 1
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Stanford University

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

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Cancer Research

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model
Kerem Nernekli *1, Dilyana B. Mangarova *1, Yifeng Shi 2, Zahra Shokri Varniab 1, Edwin Chang 1, Oguz Ziya Tikenogullari 3, Laura Pisani 1, Grigory Tikhomirov 2, Gordon Wang 4, Heike E. Daldrup-Link 1
1Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Stanford University School of Medicine, 2Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, 3Department of Mechanical Engineering, Stanford University, 4Department of Psychiatry and Behavioral Sciences, Stanford University, Wu Tsai Neuroscience Institute, Stanford University

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

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