Brigham and Women's Hospital View Institution's Website 26 articles published in JoVE Biology Partial Hepatectomy in Adult Zebrafish Isaac M. Oderberg1,2, Wolfram Goessling1,2,3 1Harvard 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. Cancer Research Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland Dongxi Xiang1,2, Luwei Tao1,2, Zhe Li1,2 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. Neuroscience Optimized Management of Endovascular Treatment for Acute Ischemic Stroke Katharina Schregel1,2, Daniel Behme1, Ioannis Tsogkas1, Michael Knauth1, Ilko Maier3, André Karch4, Rafael Mikolajczyk4,5, Mathias Bähr3, Jörn Schäper6, José Hinz6, Jan Liman3, Marios-Nikos Psychogios1 1Institute of Neuroradiology, University Medical Center Goettingen, 2 The outcome of patients with acute ischemic stroke depends on swift restoration of cerebral blood flow. This protocol aims at optimizing the management of such patients by minimizing peri-procedural timings and rendering the time from hospital admission to reperfusion as short as possible. Cancer Research Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells Nouran S. Abdelfattah1, Ann Mullally1,2,3 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 DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum Anna J. Saltman*1,2, May Barakat*1, Donald M. Bryant1, Anastasia Brodovskaya1, Jessica L. Whited1 1 Using a lipophilic 1,1'-Dioctadecy-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature. Biology Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules Anaïs Aulas*1,2, Marta M. Fay*1,2, Witold Szaflarski1,2,3, Nancy Kedersha1,2, Paul Anderson1,2, Pavel Ivanov1,2,4 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 Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae Elliott J. Hagedorn1,2, Jennifer L. Cillis3, Caitlyn R. Curley3, Taylor C. Patch3, Brian Li1,2, Bradley W. Blaser1,2,7, Raquel Riquelme1,2, Leonard I. Zon1,2,4,5,6, Dhvanit I. Shah1,2,3,4,5 1Division 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. Hutcheson3,4, Megan F. Burke2, Trejeeve Martyn1, Timothy E. Thayer2, Hannah R. Shakartzi1, Mary D. Buswell1, Robert E. Tainsh1, Binglan Yu1,4, Aranya Bagchi1,4, David K. Rhee2,4, Connie Wu1,2,4, Matthias Derwall5, Emmanuel S. Buys1,4, Paul B. Yu3,4, Kenneth D. Bloch1,2,4, Elena Aikawa3,4, Donald B. Bloch1,5,6, Rajeev Malhotra2,4 1Anesthesia 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. Behavior Community-based Adapted Tango Dancing for Individuals with Parkinson's Disease and Older Adults Madeleine E. Hackney1, Kathleen McKee2 1Center for Visual and Neurocognitive Rehabilitation, Atlanta VAMC, Department of Medicine, Division of General Medicine and Geriatrics, Emory University School of Medicine, 2 Adapted tango has demonstrated efficacy for improving mobility and balance. We describe the dissemination of adapted tango teaching methods to dance instructor trainees and the implementation of adapted tango by the trainees in the community for improving mobility and balance in older adults and individuals with Parkinson’s disease. Medicine Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection Jessica R. Allegretti1, Joshua R. Korzenik1, Matthew J. Hamilton1 1 Fecal Microbiota Transplantation via colonoscopy is a safe and effective treatment for recurrent and refractory C.difficile infection. A systematic approach to patient and donor screening, preparation of stool, and delivery of the stool during the colonoscopy will maximize therapeutic success. Immunology and Infection Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice Ilias I. Siempos1,3, Hilaire C. Lam1, Yan Ding2, Mary E. Choi2, Augustine M. K. Choi1, Stefan W. Ryter1 1 Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy. Medicine Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies Michael W. Ostler1, James H. Porter1,2, Orfeu M. Buxton1,2,3,4 1Center 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. Bioengineering Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System Oren Levy1,2,3,4,5, Priya Anandakumaran1,2,3,4,5, Jessica Ngai1,2,3,4,5, Rohit Karnik6, Jeffrey M. Karp1,2,3,4,5 1 This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy. Immunology and Infection Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria Tal Danino*1, Arthur Prindle*2, Jeff Hasty2,3,4, Sangeeta Bhatia1,5,6,7,8 1Health Sciences and Technology, Massachusetts Institute of Technology, 2Department of Bioengineering, University of California, San Diego, 3Biocircuits Institute, University of California, San Diego, 4Molecular Biology Section, Division of Biological Science, University of California, San Diego, 5Broad Institute of Harvard and MIT, 6Department of Medicine, Brigham and Women's Hospital, 7Electrical Engineering and Computer Science and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 8Howard Hughes Medical Institute The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting. Neuroscience Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue Jie Lu1, Laurent C. Delli-Bovi2, Jonathan Hecht3, Rebecca Folkerth4, Volney L. Sheen1 1Department of Neurology, Beth Israel Deaconess Medical Center, 2Department of Obstetrics and Gynecology, Brigham and Women's Hospital, 3Department of Pathology, Beth Israel Deaconess Medical Center, 4Department of Pathology, Division of Neuropathology, Brigham and Women's Hospital A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy. Biology Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells Susan Fotheringham1,2, Keren Levanon1,2,3, Ronny Drapkin1,2,4 1Department 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. Immunology and Infection Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade Jeff K. Davies1,2, Christine M. Barbon1, Annie R. Voskertchian1, Lee M. Nadler1,2, Eva C. Guinan3,4 1Medical Oncology, Dana Farber Cancer Institute, 2Department of Medicine, Brigham and Womens Hospital, 3Pediatric Oncology, Dana Farber Cancer Institute, 4Division of Hematology/Oncology, Children’s Hospital Boston This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation. Biology Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging Zhong Yang1, Qing Zeng2, Zhiyuan Ma1, Yaming Wang1, Xiaoyin Xu2 1Department of Anesthesia, Brigham and Woman's Hospital, 2Department of Radiology, Brigham and Woman's Hospital We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well. Biology Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium Georg Wiese1, Steven R. Barthel2, Charles J. Dimitroff2 1Department of Dermatology, Brigham and Women's Hospital, 2Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces. Biology Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM Kelly Morgan1, Michael Kharas1, Elaine Dzierzak2, D. Gary Gilliland1,3 1Department of Hematology and Oncology, Brigham and Women's Hospital and Harvard Medical School, 2Department of Cell Biology and Genetics, Erasmus University Medical Center, 3Department of Medicine, Howard Hughes Medical Institute, Brigham and Women's Hospital and Harvard Medical School This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells. Biology Title Cell Encapsulation by Droplets Sangjun Moon1,2, Pei-Ann Lin1,2, Hasan Onur Keles1,2, Seung-Schick Yoo3, Utkan Demirci1,2,4 1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's, Harvard Medical School, 2Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's Hospital, 3Brigham and Women's Hospital, Harvard Medical School, 4Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital Biology CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV Sang Jun Moon1, Richard Lin2, Utkan Demirci1 1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's Hospital, 2Massachusetts Institute of Technology Biology Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools Utkan Demirci1 1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's Hospital Biology A Microfluidic Device with Groove Patterns for Studying Cellular Behavior Bong Geun Chung1, Amir Manbachi1, Ali Khademhosseini1 1Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock. Biology A Gradient-generating Microfluidic Device for Cell Biology Bong Geun Chung1, Amir Manbachi1, Wajeeh Saadi1, Francis Lin1, Noo Li Jeon1, Ali Khademhosseini1 1Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis. Biology Experimental Approaches to Tissue Engineering Ali Khademhosseini1 1Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital
