Harvard Medical School

110 articles published in JoVE

• Biology

  
  Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites Kuan-Ting Huang¹, Phillippe Ly¹, Jesse R. Poganik², Saba Parvez³, Marcus J. C. Long⁴, Yimon Aye^{1 1}Swiss Federal Institute of Technology Lausanne (EPFL), ²Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, ³Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, ⁴University of Lausanne (UNIL) Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

• Biology

  
  Effective Rapid Blood Perfusion in Xenopus Rachael A. Jonas-Closs¹, Leonid Peshkin^{1 1}Department of Systems Biology, Harvard Medical School Presented here is an effective rapid blood perfusion protocol to prepare tissue samples from African clawed frogs for transcriptomics and proteomics studies.

• Neuroscience

  
  Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research Lena Sophie Koch¹, David Choy Buentello^2,3, Kerensa Broersen^{1 1}Department of Applied Stem Cell Technologies, TechMed Centre Enschede, University of Twente, ²Centro de Biotecnología-FEMSA, Tecnologico de Monterrey, ³Department of Genetics, Harvard Medical School The present protocol provides a step-by-step procedure for the reproducible generation, maintenance, and aging of cerebral organoids derived from human-induced pluripotent stem cells (iPSCs). This method enables culturing and maturing cerebral organoids for extended periods, which facilitates the modeling of processes involved in brain aging and age-related pathogenesis.

• Biology

  
  Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System Tadataka Tsuji¹, Yang Zhang¹, Yu-Hua Tseng^{1 1}Section on Integrative Physiology and Metabolism, Research Division, Joslin Diabetes Center, Harvard Medical School In this protocol, we describe the technical procedures to generate brown adipose tissue (BAT)-specific knockout mice leveraging a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The described steps include the design of the sgRNAs, the preparation of the AAV-sgRNA particles, and the microinjection of AAV into the BAT lobes.

• Immunology and Infection

  
  A Novel Method to Determine the Longitudinal Antibacterial Activity of Drug-Eluting Materials Amita Sekar^1,2, Sashank Lekkala¹, Ebru Oral^{1,2 1}Harris Orthopaedics Laboratory, Massachusetts General Hospital, ²Department of Orthopaedic Surgery, Harvard Medical School Here, we present a protocol to evaluate the antibacterial efficacy of an antibiotic-eluting polymer to simulate prophylactic clinical application by using a commercially available real-time ATP-based luminescent microbial viability assay. This method enables the monitoring of the longitudinal activity of drug-eluting materials and can be widely adapted to test anti-microbial drug delivery platforms.

• Biology

  
  Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry Tessa C. Fitch*^1,2, Scott I. Frank*¹, Yutong Kelly Li¹, Magali Saint-Geniez^1,2, Leo A. Kim^1,2, Daisy Y. Shu^{1,2 1}Schepens Eye Research Institute of Mass. Eye and Ear, ²Department of Ophthalmology, Harvard Medical School The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

• Immunology and Infection

  
  The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model Yanis Berkane*^1,2,3,5, Austin Alana Shamlou*^1,2,4,5, Jose Reyes^1,2, Hyshem H. Lancia^1,2,5, Irina Filz von Reiterdank^1,2,5,6,7, Nicolas Bertheuil³, Basak E. Uygun^2,5,6, Korkut Uygun^1,2,5,6, William G. Austen Jr.^2,4, Curtis L. Cetrulo Jr.^1,2,4,5, Mark A. Randolph^1,2,4,5, Alexandre G. Lellouch^{1,2,5,6,8 1}Vascularized Composite Allotransplantation Laboratory, Massachusetts General Hospital, ²Harvard Medical School, ³Department of Plastic, Reconstructive and Aesthetic Surgery, Rennes University Hospital Center (CHU de Rennes), University of Rennes 1, ⁴Plastic Surgery Research Laboratory, Massachusetts General Hospital, ⁵ This protocol describes harvesting, suturing, and monitoring fasciocutaneous flaps in rats that allow for good visualization and manipulation of blood flow through the superficial inferior epigastric vessels by means of clamping and ligating the femoral vessels. This is critical for studies involving ischemic preconditioning.

• Biochemistry

  
  Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells Sarah-Luisa Dülk¹, Silvia Rouskin^{1 1}Department of Microbiology, Harvard Medical School The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

• Biology

  
  Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice Taylor Stewart¹, Kevin Bode², Stephan Kissler², Peng Yi^{1 1}Section for Islet Cell and Regenerative Biology, Joslin Diabetes Center, Harvard Medical School, ²Section for Immunology, Joslin Diabetes Center, Harvard Medical School This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

• Medicine

  
  Investigation of the Electrophysiological and Thermographic Safety Parameters of Surgical Energy Devices During Thyroid and Parathyroid Surgery in a Porcine Model Hsin-Yi Tseng¹, Tzu-Yen Huang¹, Jia Joanna Wang^1,2, Yi-Chu Lin¹, I-Cheng Lu³, Feng-Yu Chiang⁴, Gianlorenzo Dionigi^5,6, Gregory W. Randolph⁷, Che-Wei Wu^{1,8 1}Department of Otorhinolaryngology-Head and Neck Surgery, International Thyroid Surgery Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, ²Department of Otolaryngology-Head and Neck Surgery, Kaohsiung Municipal Siaogang Hospital, Kaohsiung Medical University, ³Department of Anesthesiology, Kaohsiung Municipal Siaogang Hospital, Kaohsiung Medical University, ⁴Department of Otolaryngology-Head and Neck Surgery, E-Da Hospital, I-Shou University, ⁵Division of General Surgery, Endocrine Surgery Section, Istituto Auxologico Italiano (IRCCS), ⁶Department of Pathophysiology and Transplantation, Faculty of Medicine and Surgery, University of Milan, ⁷Division of Thyroid and Parathyroid Endocrine Surgery, Department of Otolaryngology-Head and Neck Surgery, Massachusetts Eye and Ear Infirmary, Harvard Medical School, ⁸Center for Liquid Biopsy and Cohort Research, and Faculty of Medicine, College of Medicine, Kaohsiung Medical University The safe application of newly developed surgical energy devices in thyroid/parathyroid surgery attracts the attention of surgeons. Animal experimental models can avoid unnecessary trials and errors in human surgery. This report aims to demonstrate electrophysiological and thermographic methods to evaluate the safety parameters of SEDs in thyroid/parathyroid surgery.

• Neuroscience

  
  Measuring Contralateral Silent Period Induced by Single-Pulse Transcranial Magnetic Stimulation to Investigate M1 Corticospinal Inhibition Ingrid Rebello-Sanchez¹, Joao Parente¹, Kevin Pacheco-Barrios^1,2, Anna Marduy¹, Danielle Carolina Pimenta¹, Daniel Lima¹, Eric Slawka¹, Alejandra Cardenas-Rojas¹, Gleysson Rodrigues Rosa¹, Kamran Nazim³, Abhishek Datta^3,4, Felipe Fregni^{1 1}Neuromodulation Center and Center for Clinical Research Learning, Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School, ²Universidad de Investigación para la Generación y Síntesis de Evidencia en Salud, Universidad San Ignacio de Loyola, ³Research and Development, Soterix Medical, ⁴City College of New York Contralateral silent period (cSP) assessment is a promising biomarker to index cortical excitability and treatment response. We demonstrate a protocol to assess cSP intended for studying M1 corticospinal inhibition of upper and lower limbs.

• Medicine

  
  Intraoperative Ultrasound in Spinal Surgery Melissa M.J. Chua¹, Viren S. Vasudeva¹, Yi Lu^{1 1}Brigham and Women's Hospital, Harvard Medical School Here, we present a protocol on the use of intraoperative ultrasound in spinal surgery, particularly in cases of intradural lesions and lesions in the ventral spinal canal when using a posterior approach.

• Medicine

  
  Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability Kiran Bora*¹, Zhongxiao Wang*¹, Felix Yemanyi¹, Meenakshi Maurya¹, Alexandra K. Blomfield¹, Yohei Tomita¹, Jing Chen^{1 1}Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

• Neuroscience

  
  Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain Gabriel S. Jensen^1,2, Jake W. Willows², David T. Breault³, Kristy L. Townsend^{1,2 1}Graduate School of Biomedical Science and Engineering, University of Maine, ²Department of Neurosurgery, Ohio State University Wexner Medical Center, ³Boston Children’s Hospital, Harvard Medical School The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

• Bioengineering

  
  Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology Sheng-Chang Luo^1,2, Ying Wang³, Ranjith Kumar Kankala^1,2, Yu Shrike Zhang⁴, Ai-Zheng Chen^{1,2 1}Institute of Biomaterials and Tissue Engineering, Huaqiao University, ²Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University, ³Affiliated Dongguan Hospital, Southern Medical University, ⁴Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening.

• Developmental Biology

  
  A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo Wei Zhang*¹, Xinjuan Lei*¹, Xin Zhou*^2,3, Boling He¹, Liqin Xiao¹, Huimin Yue¹, Shulin Wang¹, Yuting Sun¹, Yajun Wu¹, Liyang Wang^1,4, George Ghartey-Kwansah¹, Odell D. Jones⁵, Joseph L. Bryant⁶, MengMeng Xu⁷, Jianjie Ma³, Xuehon Xu^{1 1}National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China/CGDB, Shaanxi Normal University College of Life Sciences, ² Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

• Biochemistry

  
  Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy Nandini Mani*^1,2, Michelle F. Marchan*¹, Radhika Subramanian^{1,2 1}Molecular Biology, Massachusetts General Hospital, ²Department of Genetics, Harvard Medical School Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

• Medicine

  
  Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds Raffaele Di Fenza^1,3, Binglan Yu^1,3, Ryan W. Carroll^2,3, Lorenzo Berra^{1,3 1}Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, ²Department of Pediatrics, Massachusetts General Hospital, ³Harvard Medical School Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

• Neuroscience

  
  Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice Krutika Joshi¹, Alana Kirby¹, Jianguo Niu¹, Veronique VanderHorst^{1 1}Beth Israel Deaconess Medical Center, Harvard Medical School Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

• Biology

  
  Isolation of Human Neutrophils from Whole Blood and Buffy Coats Alan Y. Hsu¹, Zhicheng Peng¹, Hongbo Luo¹, Fabien Loison^{1,2 1}Department of Pathology, Harvard Medical School, Department of Lab Medicine, Children’s Hospital Boston, and Dana-Farber/Harvard Cancer Center, ²Department of Microbiology, Faculty of Science, Mahidol University This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

• Immunology and Infection

  
  Conjunctival Commensal Isolation and Identification in Mice Kirsten Smith-Page¹, Abirami Kugadas¹, Tiffany Lin¹, Mary Delaney^2,3, Lynn Bry^2,3, Mihaela Gadjeva^{1 1}Department of Medicine, Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, ²Massachusetts Host-Microbiome Center, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, ³Clinical Microbiology Laboratory, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

• Biology

  
  Partial Hepatectomy in Adult Zebrafish Isaac M. Oderberg^1,2, Wolfram Goessling^{1,2,3 1}Harvard Medical School, ² This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

• Biology

  
  Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells Blanca Chinchilla¹, Heran Getachew¹, Rosario Fernandez-Godino^{1 1}Ocular Genomics Institute of Massachusetts Eye and Ear, Harvard Medical School This protocol, which was originally reported by Fernandez-Godino et al. in 2016¹, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

• Medicine

  
  A Large Animal Model for Acute Kidney Injury by Temporary Bilateral Renal Artery Occlusion Ilias P. Doulamis¹, Alvise Guariento¹, Mossab Y. Saeed¹, Rio S. Nomoto¹, Thomas Duignan¹, Pedro J. del Nido¹, James D. McCully^{1 1}Department of Cardiac Surgery, Boston Children’s Hospital, Harvard Medical School This study presents a highly reproducible large animal model of renal ischemia-reperfusion injury in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries for 60 min and reperfusion for 24 h.

• Neuroscience

  
  Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells Sovannarath Pong^1,2,3, Paulo Lizano^1,2,3,4, Rakesh Karmacharya^{1,3,4,5 1}Center for Genomic Medicine, Massachusetts General Hospital, ²Department of Psychiatry, Beth Israel Deaconess Medical Center, ³Chemical Biology and Therapeutic Science Program, Broad Institute of MIT and Harvard, ⁴Department of Psychiatry, Harvard Medical School, ⁵Schizophrenia and Bipolar Disorder Program, McLean Hospital This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

• Biochemistry

  
  A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics Yifan Ge¹, Sivakumar Boopathy¹, Adam Smith², Luke H. Chao^{1,3 1}Department of Molecular Biology, Massachusetts General Hospital, ²Department of Chemistry, University of Akron, ³Department of Genetics, Harvard Medical School Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

• Biology

  
  An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis Yohei Tomita¹, Zhuo Shao², Bertan Cakir¹, Yumi Kotoda¹, Zhongjie Fu^1,3, Lois E.H. Smith^{1 1}Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School, ²Department of Clinical and Metabolic Genetics, Hospital for Sick Children, University of Toronto, ³Manton Center for Orphan Disease, Harvard Medical School, Boston Children's Hospital This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

• Biology

  
  A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury Hussein Atta*^1,2, Ali Hashemi Gheinani*^1,2, Amanda Wacker¹, Yaser Heshmati^3,4,5, Alex Bigger-Allen^1,6, George Lambrinos^1,2, Yao Gao^2,7, Diane R. Bielenberg^2,7, Rosalyn M. Adam^{1,2 1} The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry.

• Medicine

  
  Using an Automated Hirschberg Test App to Evaluate Ocular Alignment Gang Luo¹, Shrinivas Pundlik¹, Matteo Tomasi², Kevin Houston^{1 1}Schepens Eye Research Institute, Mass Eye & Ear, Harvard Medical School, ²EyeNexo, LLC We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

• Bioengineering

  
  Bioinspired Soft Robot with Incorporated Microelectrodes Ting Wang^1,2, Bianca Migliori^1,3, Beatrice Miccoli^1,4, Su Ryon Shin^{1 1}Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, ²School of Medicine, Jiangsu University, ³Tech4Health and Neuroscience Institutes, NYU Langone Health, ⁴Department of Electronics and Telecommunication, Politecnico di Torino A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

• Editorial

  
  Little Fish, Big Questions: A Collection of Modern Techniques for Mexican Tetra Research Misty R. Riddle¹, Clifford J. Tabin^{1 1}Genetics Department, Blavatnik Institute, Harvard Medical School

• Neuroscience

  
  Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons Debkanya Datta^1,2, Anju Vasudevan^{1,2 1}Department of Psychiatry, Harvard Medical School, ²Angiogenesis and Brain Development Laboratory, Division of Basic Neuroscience, McLean Hospital We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

• Neuroscience

  
  Functional MRI in Conjunction with a Novel MRI-compatible Hand-induced Robotic Device to Evaluate Rehabilitation of Individuals Recovering from Hand Grip Deficits Mark P. Ottensmeyer^1,2, Shasha Li^2,3,4, Gianluca De Novi^1,2, A. Aria Tzika^{2,3,4 1}Medical Device & Simulation Laboratory, Department of Radiology, Massachusetts General Hospital, ²Harvard Medical School, ³NMR Surgical Laboratory, Department of Surgery, Center for Surgery, Innovation and Bioengineering, Massachusetts General Hospital, Harvard Medical School, ⁴Athinoula A. Martinos Center of Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School We performed functional MRI using a novel MRI-compatible hand-induced robotic device to evaluate its utility for monitoring hand motor function in individuals recovering from neurological deficits.

• Medicine

  
  Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods Francisco J. Molina Estevez*¹, Tyler D. Mathews*¹, Alessandra Biffi^1,2, Marco Peviani^{1,2 1} We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

• Neuroscience

  
  Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Isl^mn:GFP Transgenic Mice Ryosuke Fujiki^1,2,3,4,9, Joun Y. Lee^1,2,10, Julie A. Jurgens^1,2,3,7, Mary C. Whitman^2,5,6, Elizabeth C. Engle^{1,2,3,4,5,6,7,8 1} This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

• Immunology and Infection

  
  Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice Xiaofang Xiong¹, Fengyang Lei², Mohammad Haque¹, Jianxun Song^{1 1}Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, ²Department of Ophthalmology, Harvard Medical School Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

• Biochemistry

  
  Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation Emily A. Brown^1,2, Steven C. Neier^3,4, Claudia Neuhauser⁵, Adam G. Schrum^6,7,8, Stephen E.P. Smith^{1,2,9 1} Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

• Bioengineering

  
  Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules Mohammed Mahamdeh^1,2, Jonathon Howard^{1 1}Department of Molecular Biophysics and Biochemistry, Yale University, ²Harvard Medical School, Harvard University This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

• Neuroscience

  
  Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth Mary C. Whitman^1,2,3, Jessica L. Bell^1,3, Elaine H. Nguyen^1,3, Elizabeth C. Engle^{1,2,3,4,5,6 1} An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 Isl^MN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

• Immunology and Infection

  
  Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas Saltanat Ualiyeva¹, Eri Yoshimoto¹, Nora A. Barrett¹, Lora G. Bankova^{1 1}Division of Rheumatology, Allergy and Immunology, Brigham and Women's Hospital, Harvard Medical School Brush cells are rare cholinergic chemosensory epithelial cells found in the naïve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

• Cancer Research

  
  Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland Dongxi Xiang^1,2, Luwei Tao^1,2, Zhe Li^{1,2 1} The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

• Medicine

  
  Assessment and Characterization of Hyaloid Vessels in Mice Zhongxiao Wang¹, Chi-Hsiu Liu¹, Shuo Huang¹, Jing Chen^{1,2 1}Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School, ²The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

• Developmental Biology

  
  Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling Kana Ishimatsu¹, Anna Cha¹, Zach M. Collins¹, Sean G. Megason^{1 1}Department of Systems Biology, Harvard Medical School Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change.

• Neuroscience

  
  Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain Faddi G. Saleh Velez^1,2, Camila B. Pinto^1,3, Emma S. Bailin⁴, Marionna Münger¹, Andrew Ellison⁵, Beatriz T. Costa¹, David Crandell⁶, Nadia Bolognini^7,8, Lotfi B. Merabet⁴, Felipe Fregni^{1 1}Laboratory of Neuromodulation & Center for Clinical Research Learning, Department of Physical Medicine and Rehabilitation, Harvard Medical School, Spaulding Rehabilitation Hospital, ²University of Chicago Medical Center, Department of Neurology, University of Chicago, ³Department of Neuroscience and Behavior, Psychology Institute, University of Sao Paulo, ⁴The Laboratory for Visual Neuroplasticity, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, ⁵Center for Biomedical Imaging, Department of Anatomy and Neurobiology, Boston University School of Medicine, ⁶Spaulding Rehabilitation Hospital, Harvard Medical School, ⁷Department of Psychology & Milan Center for Neuroscience, University of Milano-Bicocca, ⁸Neuropsychological Laboratory, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Istituto Auxologico Italiano We present a novel combined behavioral and neuroimaging protocol employing real-time video projection for the purpose of characterizing the neural correlates associated with mirror therapy within the magnetic resonance imaging scanner environment in leg amputee subjects with phantom limb pain.

• Immunology and Infection

  
  Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method Thea Brennan-Krohn^1,2,3, James E Kirby^{1,3 1}Department of Pathology, Beth Israel Deaconess Medical Center, ² Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

• Bioengineering

  
  Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens Daniel J. Wiegand^1,2, Henry H. Lee¹, Nili Ostrov*¹, George M. Church*^{1,2 1}Department of Genetics, Harvard Medical School, ²Wyss Institute for Biologically Inspired Engineering Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

• Neuroscience

  
  In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films Skyler L. Jackman¹, Christopher H. Chen², Wade G. Regehr^{2 1}Vollum Institute, Oregon Health and Science University, ²Harvard Medical School Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

• Genetics

  
  Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry Misty Riddle¹, Brian Martineau¹, Megan Peavey¹, Clifford Tabin^{1 1}Harvard Medical School In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

• Cancer Research

  
  Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos Jianhong Lin*^1,2, Yi Hu*¹, Jian-Jun Zhao^{1 1}Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, ²Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School This manuscript describes the synthesis of a single-wall carbon nanotube (SWCNT)-conjugated MALAT1 antisense gapmer DNA oligonucleotide (SWCNT-anti-MALAT1), which demonstrates the reliable delivery of the SWCNT and the potent therapeutic effect of anti-MALAT1 in vitro and in vivo. Methods used for synthesis, modification, conjugation, and injection of SWCNT-anti-MALAT1 are described.

• Cancer Research

  
  Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches Yavuz F. Yazicioglu^1,2, Halil I. Aksoylar^1,2, Rinku Pal^1,2, Nikolaos Patsoukis^1,2, Vassiliki A. Boussiotis^{1,2 1}Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, ²Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer's Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

• Neuroscience

  
  A Protocol for Transcranial Photobiomodulation Therapy in Mice Farzad Salehpour^1,2, Luis De Taboada³, Paolo Cassano^4,5,6, Farzin Kamari¹, Javad Mahmoudi¹, Sohrab Ahmadi-Kandjani⁷, Seyed Hossein Rasta^8,9,10, Saeed Sadigh-Eteghad^{1 1}Neurosciences Research Center, Tabriz University of Medical Sciences, ²ProNeuroLIGHT LLC, ³LiteCure LLC, ⁴Department of Psychiatry, Harvard Medical School, ⁵Depression Clinical and Research Program, Department of Psychiatry, Massachusetts General Hospital, ⁶Center for Anxiety and Traumatic Stress Disorders, Department of Psychiatry, Massachusetts General Hospital, ⁷Research Institute for Applied Physics and Astronomy, University of Tabriz, ⁸Department of Medical Physics, Tabriz University of Medical Sciences, ⁹Department of Medical Bioengineering, Tabriz University of Medical Sciences, ¹⁰School of Medical Sciences, University of Aberdeen Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

• Bioengineering

  
  Apparatus for Harvesting Tissue Microcolumns Joshua Tam^1,2, William Farinelli¹, Walfre Franco^1,2, R. Rox Anderson^{1,2 1}Wellman Center for Photomedicine, Massachusetts General Hospital, ²Department of Dermatology, Harvard Medical School Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

• Immunology and Infection

  
  Listeria monocytogenes Infection of the Brain Pallab Ghosh¹, Darrren E. Higgins^{1 1}Department of Microbiology and Immunobiology, Harvard Medical School During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

• Genetics

  
  CRISPR Guide RNA Cloning for Mammalian Systems Sathiji Nageshwaran*^1,2, Alejandro Chavez*^1,2,3, Nan Cher Yeo^1,2, Xiaoge Guo^1,2, Alissa Lance-Byrne¹, Angela Tung¹, James J. Collins^1,4,5,6,7, George M. Church^{1,2 1}Wyss Institute for Biologically Inspired Engineering, Harvard University, ²Department of Genetics, Harvard Medical School, ³Department of Pathology, Massachusetts General Hospital, ⁴Institute for Medical Engineering & Science, Massachusetts Institute of Technology, ⁵Synthetic Biology Center, Massachusetts Institute of Technology, ⁶Department of Biological Engineering, Massachusetts Institute of Technology, ⁷Broad Institute Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

• Biology

  
  Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes Alexander Goedel*^1,2, Dorota M. Zawada*¹, Fangfang Zhang¹, Zhifen Chen³, Alessandra Moretti^1,2, Daniel Sinnecker^{1,2 1}Medical Department I, University Hospital Klinikum rechts der Isar, Technical University of Munich, ²German Centre for Cardiovascular Research (DZHK), Munich Heart Alliance, ³Beth Israel Deaconess Medical Center, Harvard Medical School Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

• Genetics

  
  Purification of Low-abundant Cells in the Drosophila Visual System Jing Peng¹, Ivan J. Santiago¹, Matthew Y. Pecot^{1 1}Department of Neurobiology, Harvard Medical School Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

• Medicine

  
  Home-Based Transcranial Direct Current Stimulation Device Development: An Updated Protocol Used at Home in Healthy Subjects and Fibromyalgia Patients Fabiana Carvalho^1,2, Aline Patrícia Brietzke^1,2, Assunta Gasparin^1,2, Franciele Pereira dos Santos^2,3, Rafael Vercelino⁴, Rafael Firmino Ballester^2,3, Paulo Roberto Stefani Sanches⁵, Danton Pereira da Silva Jr⁵, Iraci L. S. Torres^1,6, Felipe Fregni^7,8, Wolnei Caumo^{1,2,9,10 1}Post-Graduate Program in Medical Sciences, School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS), ²Laboratory of Pain & Neuromodulation, Hospital de Clínicas de Porto Alegre (HCPA), ³School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS), ⁴Faculdade de Desenvolvimento do Rio Grande do Sul (FADERGS), Health and Wellness School Laureate International Universities, ⁵Biomedical Engineering Department, Hospital de Clínicas de Porto Alegre (HCPA), ⁶Pharmacology Department, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul (UFRGS), ⁷Department of Physical Medicine and Rehabilitation, Harvard Medical School, ⁸Spaulding Neuromodulation Center, Spaulding Rehabilitation Hospital, ⁹Pain and Palliative Care Service, Hospital de Clínicas de Porto Alegre (HCPA), ¹⁰Department of Surgery, School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS) This study provides an updated home-based tDCS protocol that enables subjects to receive the beneficial effects of tDCS at home with an easy to use device with settings to control the use and dosage, enhancing the feasibility for long-term use at home.

• Biology

  
  Diagonal Method to Measure Synergy Among Any Number of Drugs Melike Cokol-Cakmak¹, Feray Bakan², Selim Cetiner¹, Murat Cokol^{1,2,3 1}Faculty of Engineering and Natural Sciences, Sabanci University, ²Nanotechnology Research and Application Center, Sabanci University, ³Laboratory of Systems Pharmacology, Harvard Medical School In this protocol, we describe how to make Loewe additivity-based drug interaction measurements for pairwise and three-way drug combinations.

• Immunology and Infection

  
  Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification Kourtney P. Nickerson^1,2, Christina S. Faherty^{1,2 1}Mucosal Immunology and Biology Research Center, Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, ²Department of Pediatrics, Harvard Medical School This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

• Immunology and Infection

  
  Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants Eliana Drenkard*¹, Rhianna M. Hibbler*¹, D. Alina Gutu², Alexander D. Eaton¹, Amy L. Silverio¹, Frederick M. Ausubel^2,3, Bryan P. Hurley^1,4, Lael M. Yonker^{1,4 1}Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital, ²Department of Molecular Biology, Massachusetts General Hospital, ³Department of Genetics, Harvard Medical School, ⁴Department of Pediatrics, Harvard Medical School Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

• Cancer Research

  
  Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells Nouran S. Abdelfattah¹, Ann Mullally^{1,2,3 1} Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

• Biology

  
  Pulling Membrane Nanotubes from Giant Unilamellar Vesicles Coline Prévost*^1,2,3, Feng-Ching Tsai*^1,4, Patricia Bassereau^1,4, Mijo Simunovic^{1,5 1}Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, ²Department of Genetics and Complex Diseases, T. H. Chan School of Public Health, Harvard Medical School, ³Department of Cell Biology, Harvard Medical School, ⁴Sorbonne Universités, UPMC University Paris 06, ⁵Center for Studies in Physics and Biology, The Rockefeller University Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

• Behavior

  
  Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain Verónica Lamas^1,2, Sheila Estévez¹, Marianni Pernía¹, Ignacio Plaza¹, Miguel A. Merchán^{1 1}Institute of Neuroscience of Castilla y León, University of Salamanca, ²Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Department of Otolaryngology, Harvard Medical School We describe a method for the stereotactically-guided location, exposure, and ablation of the auditory cortex in rats. The localization of the ablation is assessed using a coordinate map postmortem.

• Developmental Biology

  
  The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging Nan Zhang^1,2, Edward Membreno¹, Susan Raj¹, Hongjie Zhang^1,3, Liakot A Khan¹, Verena Gobel^{1 1}Mucosal Immunology and Biology Research Center, Developmental Biology and Genetics Core, Massachusetts General Hospital for Children, Harvard Medical School, ²College of Life Sciences, Jilin University, ³Faculty of Health Sciences, University of Macau The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

• Developmental Biology

  
  The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging Nan Zhang^1,2, Liakot A Khan¹, Edward Membreno¹, Gholamali Jafari¹, Siyang Yan¹, Hongjie Zhang^1,3, Verena Gobel^{1 1}Mucosal Immunology and Biology Research Center, Developmental Biology and Genetics Core, Massachusetts General Hospital, Harvard Medical School, ²College of Life Sciences, Jilin University, ³Faculty of Health Sciences, University of Macau The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

• Bioengineering

  
  Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids Yu Shrike Zhang*¹, Qingmeng Pi*^1,2, Anne Metje van Genderen^{1,3 1}Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, ²Department of Plastic and Reconstructive Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, ³Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

• Cancer Research

  
  A Unified Methodological Framework for Vestibular Schwannoma Research Lukas D. Landegger*^1,2,3, Jessica E. Sagers*^1,4, Sonam Dilwali^1,4, Takeshi Fujita^1,2, Mehmet I. Sahin^1,2, Konstantina M. Stankovic^{1,2,4 1}Eaton Peabody Laboratories, Department of Otolaryngology, Massachusetts Eye and Ear, ²Department of Otolaryngology, Harvard Medical School, ³Department of Otolaryngology, Vienna General Hospital, Medical University of Vienna, ⁴Program in Speech and Hearing Bioscience and Technology, Harvard Medical School The goal of this protocol is to outline the collection and processing of human surgical samples for multiple downstream applications in vestibular schwannoma and Schwann cell research.

• Medicine

  
  Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research Lukas D. Landegger^1,2,3, Sonam Dilwali^1,4, Konstantina M. Stankovic^{1,2,4 1}Eaton Peabody Laboratories, Department of Otolaryngology, Massachusetts Eye and Ear, ²Department of Otolaryngology, Harvard Medical School, ³Department of Otolaryngology, Vienna General Hospital, Medical University of Vienna, ⁴Harvard Program in Speech and Hearing Bioscience and Technology The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

• Biology

  
  Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules Anaïs Aulas*^1,2, Marta M. Fay*^1,2, Witold Szaflarski^1,2,3, Nancy Kedersha^1,2, Paul Anderson^1,2, Pavel Ivanov^{1,2,4 1} Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

• Developmental Biology

  
  A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening Charles A. C. Williams¹, Nathanael S. Gray^2,3, Greg M. Findlay^{1 1}The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, ²Department of Cancer Biology, Dana-Farber Cancer Institute, ³Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the naïve-primed pluripotent transition.

• Medicine

  
  Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation Ruiye Bi^1,2, Yi Fan^2,3, En Luo^2,4, Quan Yuan^2,4, Michael Mannstadt^{1 1}Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, ²West China School of Stomatology, Sichuan University, ³Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, ⁴State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor.

• Genetics

  
  Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins Alfredo J. Hernandez¹, Charles C. Richardson^{1 1}Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

• Genetics

  
  Detection of Copy Number Alterations Using Single Cell Sequencing Kristin A. Knouse^1,2,3, Jie Wu⁴, Austin Hendricks^{5 1}Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, ²Howard Hughes Medical Institute, ³Division of Health Sciences and Technology, Harvard Medical School, ⁴The Barbara K. Ostrom (1978) Bioinformatics and Computing Facility in the Swanson Biotechnology Center, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, ⁵BioMicro Center, Department of Biology, Massachusetts Institute of Technology Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

• Neuroscience

  
  Measuring and Altering Mating Drive in Male Drosophila melanogaster Christine L. Boutros*¹, Lauren E. Miner*¹, Ofer Mazor^2,3, Stephen X. Zhang^{3 1} This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

• Genetics

  
  Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts Jian Ding^1,2, Zhi-Qiang Lin^1,2, Jian-Ming Jiang^3,4, Christine E. Seidman^3,4, Jonathan G. Seidman^3,4, William T. Pu^1,2, Da-Zhi Wang^{1,2 1} In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

• Developmental Biology

  
  Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy Haochuan Zhang*^1,2,3, JunGang Huang*^2,3,4, Xin Liu^2,3, Ping Zhu⁴, Zhongrong Li¹, Xue Li^{2,3 1}Department of Pediatric Surgery, The Second Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, ² We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens.

• Medicine

  
  A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy Mouhsin M. Shafi^1,2,3, Susan Whitfield-Gabrieli⁴, Catherine J. Chu^1,5, Alvaro Pascual-Leone^1,2,3, Bernard S. Chang^{1,2 1}Department of Neurology, Harvard Medical School, ²Department of Neurology, Beth Israel Deaconess Medical Center, ³Berenson-Allen Center for Noninvasive Brain Stimulation, Beth Israel Deaconess Medical Center, ⁴Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, ⁵Department of Neurology, Massachusetts General Hospital Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

• Developmental Biology

  
  Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions Melinda L. Koyle¹, Madeline Veloz¹, Alec M. Judd¹, Adam C.-N. Wong^2,4, Peter D. Newell^2,5, Angela E. Douglas^2,3, John M. Chaston^{1,2 1}Department of Plant and Wildlife Sciences, Brigham Young University, ²Department of Entomology, Cornell University, ³Department of Molecular Biology and Genetics, Cornell University, ⁴Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, ⁵Biological Sciences, SUNY Oswego A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

• Developmental Biology

  
  Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae Elliott J. Hagedorn^1,2, Jennifer L. Cillis³, Caitlyn R. Curley³, Taylor C. Patch³, Brian Li^1,2, Bradley W. Blaser^1,2,7, Raquel Riquelme^1,2, Leonard I. Zon^1,2,4,5,6, Dhvanit I. Shah^{1,2,3,4,5 1}Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, ⁴Harvard Stem Cell Institute, ⁵Broad Institute of Massachusetts Institute of Technology, ⁶Howard Hughes Medical Institute, ⁷Division of Hematologic Malignancies, Dana-Farber Cancer Institute This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

• Medicine

  
  Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation Caitlin O'Rourke*¹, Georgia Shelton*^1,2, Joshua D. Hutcheson^3,4, Megan F. Burke², Trejeeve Martyn¹, Timothy E. Thayer², Hannah R. Shakartzi¹, Mary D. Buswell¹, Robert E. Tainsh¹, Binglan Yu^1,4, Aranya Bagchi^1,4, David K. Rhee^2,4, Connie Wu^1,2,4, Matthias Derwall⁵, Emmanuel S. Buys^1,4, Paul B. Yu^3,4, Kenneth D. Bloch^1,2,4, Elena Aikawa^3,4, Donald B. Bloch^1,5,6, Rajeev Malhotra^{2,4 1}Anesthesia Center for Critical Care Research of the Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, ²Cardiovascular Research Center and Cardiology Division of the Department of Medicine, Massachusetts General Hospital, ³ Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

• Immunology and Infection

  
  Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions Achille Broggi^1,2, Clara Cigni¹, Ivan Zanoni^1,2,3, Francesca Granucci^{1,3 1}Department of Biotechnology and Biosciences, University of Milano-Bicocca, ²Boston Children's Hospital, Division of Gastroenterology, Harvard Medical School, ³Humanitas Clinical and Research Center The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

• Immunology and Infection

  
  Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C) Ke-Huan Chow*¹, Zihao Yan*^1,2, Wei-Li Wu^{1 1}Division of Biology and Biological Engineering, California Institute of Technology, ²Harvard Medical School Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

• Neuroscience

  
  A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster Severine Trannoy¹, Budhaditya Chowdhury¹, Edward A. Kravitz^{1 1}Department of Neurobiology, Harvard Medical School During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong "loser" effects.

• Medicine

  
  Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats Lauren L. Jantzie^1,2, Jesse L. Winer³, Jessie R. Maxwell¹, Lindsay A.S. Chan³, Shenandoah Robinson^{3,4 1}Department of Pediatrics, University of New Mexico, ²Department of Neurosciences, University of New Mexico, ³ Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

• Developmental Biology

  
  Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage Lucie J. Rochard*¹, Irving T.C. Ling*¹, Yawei Kong¹, Eric C. Liao^{1 1}Massachusetts General Hospital, Center for Regenerative Medicine, Harvard Medical School Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel’s cartilage.

• Developmental Biology

  
  Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury Caramai N. Kamei¹, Yan Liu^1,2, Iain A. Drummond^{1,3 1}Nephrology Division, Department of Medicine, Massachusetts General Hospital, ²Basic Sciences Division, Fred Hutchinson Cancer Research Center, ³Department of Genetics, Harvard Medical School Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

• Chemistry

  
  Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles Bryan Wei¹, Michelle K. Vhudzijena², Joanna Robaszewski², Peng Yin^{2,3 1}Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, ²Wyss Institute for Biologically Inspired Engineering, Harvard University, ³Department of Systems Biology, Harvard Medical School DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

• Behavior

  
  Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats Lisa Y. Maeng^1,2, Kara K. Cover¹, Aaron J. Landau¹, Mohammed R. Milad^1,2, Kelimer Lebron-Milad^{1,2 1}Department of Psychiatry, Massachusetts General Hospital, ²Harvard Medical School Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

• Medicine

  
  Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres Shannon Bunker¹, Joanna Holeniewska¹, Sauparnika Vijay², Annegret Dahlmann-Noor^2,3, Peng Khaw^2,4, Yin-Shan Ng⁵, David Shima^1,6, Richard Foxton^{1 1}Ocular Biology and Therapeutics, University College London Institute of Ophthalmology, ²University College London Institue of Ophthalmology, ³Moorfields Eye Hospital, ⁴NIHR Biomedical Research Centre, Moorfields Eye Hospital, ⁵Schepens Eye Research Institute, Harvard Medical School, ⁶Hoffman-La Roche We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

• Neuroscience

  
  Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway Elliott D. Kozin*¹, Keith N. Darrow*², Ariel E. Hight*¹, Ashton E. Lehmann¹, Alyson B. Kaplan¹, M. Christian Brown¹, Daniel J. Lee^{1 1}Department of Otology and Laryngology, Harvard Medical School, ²Department of Communication Sciences and Disorders, Worcester State University The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

• Biology

  
  Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 Daniel E. Bauer*^1,2,3, Matthew C. Canver*¹, Stuart H. Orkin^{1,2,3,4 1}Harvard Medical School, ² CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

• Bioengineering

  
  Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay Lama Mulki*^1,2, J. Harry Sweigard*¹, Kip M. Connor^{1 1}Angiogenesis Laboratory, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye & Ear Infirmary, ² Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

• Biology

  
  Assessing Cerebral Autoregulation via Oscillatory Lower Body Negative Pressure and Projection Pursuit Regression J. Andrew Taylor^1,2, Can Ozan Tan^1,2, J. W. Hamner^{2 1}Department of Physical Medicine and Rehabilitation, Harvard Medical School, ²Cardiovascular Research Laboratory, Spaulding Hospital Cambridge Cerebral perfusion is maintained across a range of pressures via cerebral autoregulation. However, characterizing autoregulation requires prominent pressure fluctuations at regulated frequencies. The described protocol will show how oscillatory lower body negative pressure can generate pressure fluctuations to provide data for projection pursuit regression for quantification of the autoregulatory curve.

• Biology

  
  Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology William S. Phipps*¹, Zhizhong Yin*¹, Candice Bae¹, Julia Z. Sharpe¹, Andrew M. Bishara², Emily S. Nelson³, Aaron S. Weaver³, Daniel Brown⁴, Terri L. McKay³, DeVon Griffin³, Eugene Y. Chan^{1 1}DNA Medicine Institute, ²Harvard Medical School, ³NASA Glenn Research Center, ⁴ZIN Technologies Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

• Neuroscience

  
  The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye Amy E. Birsner*¹, Ofra Benny*², Robert J. D'Amato^{1,3 1} The protocol describes the corneal micropocket assay as developed in mice.

• Medicine

  
  Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice Richie E. Kohman¹, Xue Han¹, Benjamin S. Bleier^{2 1}Department 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.

• Bioengineering

  
  Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood Caroline N. Jones*^1,2,3, Anh N. Hoang*^1,2,3, Laurie Dimisko¹, Bashar Hamza¹, Joseph Martel^1,4, Daniel Irimia^{1,2,3 1}The BioMEMS Resource Center, Department of Surgery, Massachusetts General Hospital, ²Harvard Medical School, ³Shriners Burns Hospital, ⁴Harvard University School of Engineering and Applied Sciences This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

• Bioengineering

  
  Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration Lindsay M. Thomson¹, Brian D. Polizzotti¹, Frances X. McGowan², John N. Kheir^{1 1}Department of Cardiology, Boston Children's Hospital, Harvard Medical School, ²Department of Anesthesiology and Critical Care, Children's Hospital of Philadelphia, University of Pennsylvania We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

• Medicine

  
  Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair Dongsu Park¹, Joel A. Spencer², Charles P. Lin², David T. Scadden^{1 1}Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Stem Cell Institute, ²Wellman Center for Photomedicine and Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

• Environment

  
  Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun Matthew D. Mattozzi^1,2, Mathias J. Voges^1,2,3, Pamela A. Silver^1,2, Jeffrey C. Way^{1,2 1}Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard University, ²Department of Systems Biology, Harvard Medical School, ³Department of Biotechnology, Delft University of Technology This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

• Biology

  
  Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions Chi-Iou Lin¹, Chun-Yee Lau¹, Dan Li¹, Shou-Ching Jaminet^{1 1}Center for Vascular Biology Research, Department of of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School Nanopodia are thin but fragile membrane channels that extend up to 100 μm from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 °C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

• Neuroscience

  
  Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans Limin Hao^1,2, Edgar A. Buttner^{1,2 1}Department of Psychiatry, Harvard Medical School, ²Mailman Research Center, McLean Hospital Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

• 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 Cheng¹, Michael Bauer¹, Soeun Ngoy¹, Yiling Qiu¹, Jian Guan¹, Shikha Mishra¹, Christopher Mbah¹, Ronglih Liao^{1 1}Cardiac 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.

• Medicine

  
  Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies Michael W. Ostler¹, James H. Porter^1,2, Orfeu M. Buxton^{1,2,3,4 1}Center for Population and Development Studies, Harvard School of Public Health, ² Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates and analyzed for a variety of biomarkers.

• Neuroscience

  
  Isolation and Culture of Endothelial Cells from the Embryonic Forebrain Peeyush Kumar T.^1,2, Anju Vasudevan^{1,2 1}Department of Psychiatry, Harvard Medical School, ²Angiogenesis and Brain Development Laboratory, Division of Basic Neuroscience, McLean Hospital This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

• Bioengineering

  
  An Improved Method for the Preparation of Type I Collagen From Skin Christina A. Pacak^1,2, Allison A. MacKay¹, Douglas B. Cowan^{1,2 1} Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

• Medicine

  
  Ultrasonic Assessment of Myocardial Microstructure Pranoti Hiremath¹, Michael Bauer², Hui-Wen Cheng², Kazumasa Unno², Ronglih Liao², Susan Cheng^{2 1}Harvard Medical School, ² 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.

• Bioengineering

  
  Stress-induced Antibiotic Susceptibility Testing on a Chip Maxim Kalashnikov¹, Jennifer Campbell¹, Jean C. Lee², Andre Sharon^1,3, Alexis F. Sauer-Budge^{1,4 1}Fraunhofer 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.

• Immunology and Infection

  
  In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration Mark E. Kusek^1,2,3, Michael A. Pazos^1,3, Waheed Pirzai³, Bryan P. Hurley^{1,3 1}Department of Pediatrics, Harvard Medical School, ²Department of Pediatric Gastroenterology, MGH for Children, ³Mucosal Immunology and Biology Research Center, Massachusetts General Hospital Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease. An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

• Biology

  
  Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells Susan Fotheringham^1,2, Keren Levanon^1,2,3, Ronny Drapkin^{1,2,4 1}Department of Medical Oncology, Dana-Farber Cancer Institute, ²Harvard Medical School, Boston, MA, ³Sheba Cancer Research Center, Chaim Sheba Medical Center, ⁴Department of Pathology, Brigham and Women's Hospital The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.