Massachusetts Institute of Technology View Institution's Website 81 articles published in JoVE Cancer Research Venous Thrombosis Assay in a Mouse Model of Cancer Saran Lotfollahzadeh1, Xiaosheng Yang1, David Jasen Wu Wong1, Jingyan Han2, Francesca Seta2, Suvranu Ganguli3, Asha Jose1, Katya Ravid4,5, Vipul C. Chitalia1,6,7,8 1Renal Section, Department of Medicine, Chobanian and Avedisian School of Medicine, Boston University, 2Vascular Biology Section, Department of Medicine, Chobanian and Avedisian School of Medicine, Boston University, 3Division of Interventional Radiology, Department of Radiology, Chobanian and Avedisian School of Medicine, Boston University, 4Department of Medicine, Whitaker Cardiovascular Institute, Chobanian and Avedisian School of Medicine, Boston University, 5Department of Biochemistry and Cell Biology, Chobanian and Avedisian School of Medicine, Boston University, 6Veterans Affairs Boston Healthcare System, 7Institute of Medical Engineering and Sciences, Massachusetts Institute of Technology, 8Center of Cross-Organ Vascular Pathology, Department of Medicine, Chobanian and Avedisian School of Medicine, Boston University This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis. Bioengineering CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics Audrey E. Van Heest1, Feiyang Deng1, Renee T. Zhao2, Nour Saida Harzallah2,3, Heather E. Fleming3,4, Sangeeta N. Bhatia2,3,4,5,6,7,8, Liangliang Hao1,2,3 1Department of Biomedical Engineering, Boston University, 2Institute for Medical Engineering and Science, Massachusetts Institute of Technology, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Howard Hughes Medical Institute, 5Broad Institute of Massachusetts Institute of Technology and Harvard, 6Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 7Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 8 This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities. Biochemistry Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination Andrew V. Grassetti*1, Mira B. May*1, Joseph H. Davis1,2 1Department of Biology, Massachusetts Institute of Technology, 2Program in Computational and Systems Biology, Massachusetts Institute of Technology The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation. Engineering Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection Fangzhou Xia1, Kamal Youcef-Toumi1, Thomas Sattel2, Eberhard Manske3, Ivo W. Rangelow4,5 1Mechatronics Research Lab, Department of Mechanical Engineering, Massachusetts Institute of Technology, 2Mechatronics Group, Department of Mechanical Engineering, Ilmenau University of Technology, 3Production and Precision Measurement Technology Group, Institute of Process Measurement and Sensor Technology, Ilmenau University of Technology, 4Nanoscale Systems Group, Institute of Process Measurement and Sensor Technology, Ilmenau University of Technology, 5nano analytik GmbH Large-scale sample inspection with nanoscale resolution has a wide range of applications, especially for nanofabricated semiconductor wafers. Atomic force microscopes can be a great tool for this purpose, but are limited by their imaging speed. This work utilizes parallel active cantilever arrays in AFMs to enable high-throughput and large-scale inspections. Bioengineering Rapid Development of Cell State Identification Circuits with Poly-Transfection Noreen Wauford1, Ross Jones1,2, Charles Van De Mark3, Ron Weiss1,3 1Department of Biological Engineering, Massachusetts Institute of Technology, 2School of Biological Engineering, University of British Columbia, 3Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis. Medicine A Retrograde Implantation Approach for Peritoneal Dialysis Catheter Placement in Mice Saran Lotfollahzadeh1, Mengwei Zhang1, Marc Arthur Napoleon1, Wenqing Yin1, Josephine Orrick1, Nagla Elzind1, Austin Morrissey1, Isaac E. Sellinger1, Lauren D. Stern1, Mostafa Belghasem2, Jean M. Francis1, Vipul C. Chitalia1,3,4 1Renal Section, Department of Medicine, Boston University Aram V. Chobanian & Edward Avedisian School of Medicine, 2Department of Biomedical Science, Kaiser Permanente Bernard J. Tyson School of Medicine, 3Veterans Affairs Boston Healthcare System, 4Institute of Medical Engineering and Sciences, Massachusetts Institute of Technology This article describes modifications of a procedure to implant a peritoneal dialysis catheter in a murine model to avoid major technical issues observed with the conventional techniques. Cancer Research Longitudinal Intravital Imaging Through Clear Silicone Windows Laura Maiorino*1,2,3, Margaret Shevik*1,4,5, José M. Adrover1, Xiao Han1,6, Elias Georgas7,8, John Erby Wilkinson9, Harrison Seidner1, Leonie Foerschner1, David A. Tuveson1, Yi-Xian Qin8, Mikala Egeblad1 1Cold Spring Harbor Laboratory, 2Cold Spring Harbor Laboratory School of Biological Sciences, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Medical Scientist Training Program, School of Medicine, Stony Brook University, 5Graduate Program in Pharmacology, Stony Brook University, 6Graduate Program in Genetics, Stony Brook University, 7Graduate Program in Biomedical Engineering, Stony Brook University, 8Department of Biomedical Engineering, Stony Brook University, 9Department of Pathology, University of Michigan An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals. Biochemistry Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes Daniel Barrera1, Sierra Upton2, Megan Rauch3, Tara Notarianni4, Ki Suk Eum5, Megan Liberty6, Sarmila Venkoba Sah7, Robert Liu8, Sean Newcomer9, Linda E. May10, Emre Agbas11, Jessica Sage12, Edina Kosa7, Abdulbaki Agbas7,13 1AdventHelath, 2University of Illinois Chicago School of Public Health, 3Lincoln Memorial University, 4Cincinnati Children’s Hospital Medical Center, 7Kansas City University, 8Roblex Rex Veterans Affairs Medical Center, 9California State University San Marcos, 10East Carolina University, 11Massachusetts Institute of Technology, 12Boehringer Ingelheim Norway KS, 13Heartland Center for Mitochondrial Medicine Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria. Engineering Lumped-Parameter and Finite Element Modeling of Heart Failure with Preserved Ejection Fraction Luca Rosalia*1,2, Caglar Ozturk*1, Ellen T. Roche1,2,3 1Institute for Medical Engineering and Science, Massachusetts Institute of Technology, 2Health Science and Technology Program, Harvard/Massachusetts Institute of Technology, 3Department of Mechanical Engineering, Massachusetts Institute of Technology This work introduces two computational models of heart failure with preserved ejection fraction based on a lumped-parameter approach and finite element analysis. These models are used to evaluate the changes in the hemodynamics of the left ventricle and related vasculature induced by pressure overload and diminished ventricular compliance. Engineering Theoretical Calculation and Experimental Verification for Dislocation Reduction in Germanium Epitaxial Layers with Semicylindrical Voids on Silicon Motoki Yako1, Yasuhiko Ishikawa2, Eiji Abe1, Kazumi Wada1,3 1Department of Materials Engineering, The University of Tokyo, 2Department of Electrical and Electronic Information Engineering, Toyohashi University of Technology, 3Department of Materials Science and Engineering, Massachusetts Institute of Technology Theoretical calculation and experimental verification are proposed for a reduction of threading dislocation (TD) density in germanium epitaxial layers with semicylindrical voids on silicon. Calculations based on the interaction of TDs and surface via image force, TD measurements, and transmission electron microscope observations of TDs are presented. Chemistry Facile Synthesis of Colloidal Lead Halide Perovskite Nanoplatelets via Ligand-Assisted Reprecipitation Seung Kyun Ha1, William A Tisdale1 1Department of Chemical Engineering, Massachusetts Institute of Technology This work demonstrates facile room-temperature synthesis of colloidal quantum-confined lead halide perovskite nanoplatelets by ligand-assisted reprecipitation method. Synthesized nanoplatelets show spectrally narrow optical features and continuous spectral tunability throughout the visible range by varying the composition and thicknesses. Bioengineering High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain Ekta Makhija1, Anna Jagielska1,2, Krystyn J. Van Vliet1,2,3 1BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, CREATE, 2Department of Material Science and Engineering, Massachusetts Institute of Technology, 3Department of Biological Engineering, Massachusetts Institute of Technology Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective. Cancer Research The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma Mercedes M. Riley1,2, Pyay San1, Edwin Lok1, Kenneth D. Swanson1, Eric T. Wong1 1Brain Tumor Center and Neuro-Oncology Unit, Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, 2Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment. Engineering Design and Fabrication of an Optical Fiber Made of Water Mark L. Douvidzon1, Shai Maayani2, Leopoldo L. Martin3, Tal Carmon4 1Department of Nanoscience and Nanotechnology, Russell Berrie Nanotechnology Institute (RBNI), Technion - Israel Institute of Technology, 2Department of Material Sciences and Engineering, MIT, 3Centro de Tecnologia Nanofotónica, Universitat Politècnica de València, 4Department of Mechanical Engineering, Technion - Israel Institute of Technology This protocol describes the design and manufacture of a water bridge and its activation as a water fiber. The experiment demonstrates that capillary resonances of the water fiber modulate its optical transmission. Genetics CRISPR Guide RNA Cloning for Mammalian Systems Sathiji Nageshwaran*1,2, Alejandro Chavez*1,2,3, Nan Cher Yeo1,2, Xiaoge Guo1,2, Alissa Lance-Byrne1, Angela Tung1, James J. Collins1,4,5,6,7, George M. Church1,2 1Wyss 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. Engineering Multiscale Structures Aggregated by Imprinted Nanofibers for Functional Surfaces Yeonho Jeong*1, Seok Kim*2, Nicholas Xuanlai Fang2, Seunghang Shin1, Hyunmin Choi1, Seonjun Kim1, Sin Kwon3, Young Tae Cho1 1Department of Mechanical Engineering, Changwon National University, 2Department of Mechanical Engineering, Massachusetts Institute of Technology, 3Printed Electronics Research Team, Korea Institute of Machinery and Materials Presented is an easy method to fabricate nano-micro multiscale structures, for functional surfaces, by aggregating nanofibers fabricated using an anodic aluminum oxide filter. Biochemistry Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy Alexandra J. Machen1, Pierce T. O'Neil1, Bradley L. Pentelute2, Maria T. Villar1, Antonio Artigues1, Mark T. Fisher1 1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 2Department of Chemistry, Massachusetts Institute of Technology Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively. Biology Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling Hiroshi I. Suzuki1, Masafumi Horie2,3, Hajime Mihira4, Akira Saito2 1David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 2Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 3Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, 4Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-β in MS-1 endothelial cells. Immunology and Infection Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics Hyunryul Ryu1, Kyungyong Choi1,2, Yanyan Qu3, Taehong Kwon1,2, Janet S. Lee3,5, Jongyoon Han1,2,4 1Research Laboratory of Electronics, Massachusetts Institute of Technology, 2Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 3Department of Medicine, University of Pittsburgh, 4Department of Biological Engineering, Massachusetts Institute of Technology, 5Vascular Medicine Institute, University of Pittsburgh In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases. Engineering A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals Robert W Epps1, Kobi C Felton1, Connor W Coley2, Milad Abolhasani1 1Chemical and Biomolecular Engineering, North Carolina State University, 2Chemical Engineering, Massachusetts Institute of Technology Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space. Cancer Research Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes Ruoxi W. Wang*1, Emily MacDuffie*1, Stefano Santaguida1 1Koch Institute for Integrative Cancer Research at MIT, Department of Biology, Massachusetts Institute of Technology Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide. Behavior Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates Leah C Acker1,2,3, Erica N. Pino1,4,5, Edward S. Boyden1,6,7, Robert Desimone1,7 1McGovern Institute, Massachusetts Institute of Technology, 2Harvard-MIT Division of Heath Sciences and Technology, 3Department of Anesthesiology, Duke University Medical Center, 4Department of Biology, Massachusetts Institute of Technology, 5Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 6Media Lab and Department of Biological Engineering, Massachusetts Institute of Technology, 7Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology A protocol to build a tissue penetrating illuminator for delivering light over large volumes with minimal diameter is presented. Biochemistry A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA Kan Xiong1,2, Paul C. Blainey1,2 1Broad Institute, Massachusetts Institute of Technology and Harvard Medical School, 2Dept. of Biological Engineering, Massachusetts Institute of Technology This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA. Genetics Detection of Copy Number Alterations Using Single Cell Sequencing Kristin A. Knouse1,2,3, Jie Wu4, Austin Hendricks5 1Koch 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. Medicine A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy Mouhsin M. Shafi1,2,3, Susan Whitfield-Gabrieli4, Catherine J. Chu1,5, Alvaro Pascual-Leone1,2,3, Bernard S. Chang1,2 1Department 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 Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue James E. Baily1, William C.W. Chen2,3, Nusrat Khan4, Iain R. Murray4, Zaniah N. González Galofre4, Johnny Huard5,6, Bruno Péault4,7 1Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 2Department of Bioengineering and Orthopaedic Surgery, University of Pittsburgh, 3Research Laboratory of Electronics and Department of Biological Engineering, Massachusetts Institute of Technology, 4MRC Centre for Regenerative Medicine, University of Edinburgh, 5Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, 6Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, 7Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, David Geffen School of Medicine, University of California at Los Angeles Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium. Neuroscience Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry Elizabeth Peruski Canovic1, Bo Qing2, Aleksandar S. Mijailovic3, Anna Jagielska2, Matthew J. Whitfield2, Elyza Kelly4, Daria Turner4, Mustafa Sahin4, Krystyn J. Van Vliet1,2 1Department of Materials Science and Engineering, Massachusetts Institute of Technology, 2Department of Biological Engineering, Massachusetts Institute of Technology, 3Department of Mechanical Engineering, Massachusetts Institute of Technology, 4 We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales. Engineering Applying X-ray Imaging Crystal Spectroscopy for Use as a High Temperature Plasma Diagnostic Norman M. Cao1, Andrés M. Mier Valdivia2, John E. Rice3 1Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, 2Department of Physics, Massachusetts Institute of Technology, 3Plasma Science and Fusion Center, Massachusetts Institute of Technology X-ray spectra provide a wealth of information on high temperature plasmas. This manuscript presents the operation of a high wavelength resolution spatially imaging X-ray spectrometer used to view hydrogen- and helium-like ions of medium atomic number elements in a tokamak plasma. Engineering Electrospray Deposition of Uniform Thickness Ge23Sb7S70 and As40S60 Chalcogenide Glass Films Spencer Novak1, Pao-Tai Lin2,3, Cheng Li4, Nikolay Borodinov1, Zhaohong Han5, Corentin Monmeyran5, Neil Patel5, Qingyang Du5, Marcin Malinowski4, Sasan Fathpour4, Chatdanai Lumdee4, Chi Xu4, Pieter G. Kik4, Weiwei Deng6, Juejun Hu7, Anuradha Agarwal7, Igor Luzinov1, Kathleen Richardson4 1Department of Materials Science and Engineering, Clemson University, 2Department of Materials Science and Engineering, Texas A&M University, 3Department of Electrical and Computer Engineering, Texas A&M University, 4College of Optics and Photonics, Center for Research and Education in Optics and Lasers (CREOL), University of Central Florida, 5Department of Materials Science and Engineering, Massachusetts Institute of Technology, 6Department of Mechanical Engineering, Virginia Polytechnic Institute, 7Microphotonics Center, Massachusetts Institute of Technology A method of uniform thickness solution-derived chalcogenide glass film deposition is demonstrated using computer numerical controlled motion of a single-nozzle electrospray. Engineering Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles Xi Wei1,2, Abeer Syed1, Pan Mao3, Jongyoon Han4, Yong-Ak Song1,2 1Division of Engineering, New York University Abu Dhabi (NYUAD), 2Department of Chemical and Biomolecular Engineering, New York University Tandon School of Engineering, 3Newomics, Inc., 4Department of Electrical Engineering and Computer Science, Department of Biological Engineering, MIT We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples. Chemistry Reverse Microemulsion-mediated Synthesis of Monometallic and Bimetallic Early Transition Metal Carbide and Nitride Nanoparticles Sean T. Hunt1, Yuriy Román-Leshkov1 1Chemical Engineering, Massachusetts Institute of Technology A “removable ceramic coating method” is presented in visual format for the synthesis of non-sintered and metal-terminated monometallic and bimetallic early transition metal carbide and nitride nanoparticles with tunable sizes and crystal structures. Biology Simple Bulk Readout of Digital Nucleic Acid Quantification Assays Leanna S. Morinishi1, Paul Blainey2 1Broad Institute, 2Department of Biological Engineering, Massachusetts Institute of Technology We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy. Biology Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis Alexandra Naba1, Karl R. Clauser2, Richard O. Hynes1 1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 2Proteomics Platform, Broad Institute This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry. Engineering Making Record-efficiency SnS Solar Cells by Thermal Evaporation and Atomic Layer Deposition Rafael Jaramillo2,4, Vera Steinmann1,2, Chuanxi Yang3, Katy Hartman2,4, Rupak Chakraborty1,2, Jeremy R. Poindexter2,4, Mariela Lizet Castillo2, Roy Gordon5, Tonio Buonassisi1,2 1Department of Mechanical Engineering, Massachusetts Institute of Technology, 2Laboratory for Manufacturing and Productivity, Massachusetts Institute of Technology, 3School of Engineering and Applied Sciences, Harvard University, 4Department of Materials Science and Engineering, Massachusetts Institute of Technology, 5Department of Chemistry & Chemical Biology, Harvard University Tin sulfide (SnS) is a candidate material for Earth-abundant, non-toxic solar cells. Here, we demonstrate the fabrication procedure of the SnS solar cells employing atomic layer deposition, which yields 4.36% certified power conversion efficiency, and thermal evaporation which yields 3.88%. Biology Sampling Blood from the Lateral Tail Vein of the Rat Graham Lee1, Ki A. Goosens1,2 1McGovern Institute for Brain Research, Massachusetts Institute of Technology, 2Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness. Neuroscience In vivo Optogenetic Stimulation of the Rodent Central Nervous System Michelle M. Sidor1, Thomas J. Davidson2, Kay M. Tye3, Melissa R. Warden4, Karl Diesseroth2,5, Colleen A. McClung1 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. Bioengineering CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells Jing Ge*1, Somsak Prasongtanakij*2, David K. Wood3, David M. Weingeist1, Jessica Fessler1, Panida Navasummrit2, Mathuros Ruchirawat2, Bevin P. Engelward1 1Department of Biological Engineering, Massachusetts Institute of Technology, 2Environmental Toxicology, Chulabhorn Graduate Institute, 3Department of Biomedical Engineering, University of Minnesota We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics. Neuroscience Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice Philip M. Brunetti*1, Ralf D. Wimmer*1, Li Liang1, Joshua H. Siegle2, Jakob Voigts2, Matthew Wilson2, Michael M. Halassa1 1The Neuroscience Institute, New York University Langone Medical Center, 2Department of Brain and Cognitive Science, Massachusetts Institute of Technology Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse. Biology Facial Transplants in Xenopus laevis Embryos Laura A. Jacox*1,3, Amanda J. Dickinson*4, Hazel Sive2,3 1Biological Sciences in Dental Medicine, Harvard University, 2Biology Department, Massachusetts Institute of Technology, 3Whitehead Institute, Massachusetts Institute of Technology, 4Biology Department, Virginia Commonwealth University A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions. Neuroscience In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices Martina Sonego*1, Ya Zhou*1, Madeleine Julie Oudin2, Patrick Doherty1, Giovanna Lalli1 1Wolfson Centre for Age-Related Diseases, King's College London, 2David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking. Bioengineering Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform Armon Sharei1, Nahyun Cho1, Shirley Mao1, Emily Jackson1, Roberta Poceviciute1, Andrea Adamo1, Janet Zoldan2, Robert Langer1,2, Klavs F Jensen1 1Department of Chemical Engineering, Massachusetts Institute of Technology, 2David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications. 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. Chemistry Particles without a Box: Brush-first Synthesis of Photodegradable PEG Star Polymers under Ambient Conditions Jenny Liu1, Angela Xiaodi Gao1, Jeremiah A. Johnson1 1Department of Chemistry, Massachusetts Institute of Technology Poly(ethylene glycol) (PEG) brush-arm star polymers (BASPs) with narrow mass distributions and tunable nanoscopic sizes are synthesized in via ring opening metathesis polymerization (ROMP) of a PEG-norbornene macromonomer followed by transfer of portions of the resulting living brush initiator to vials containing varied amounts of a rigid, photo-cleavable bis-norbornene crosslinker. Biology Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology Zachary Z. Sun*1, Clarmyra A. Hayes*2, Jonghyeon Shin3, Filippo Caschera4, Richard M. Murray2, Vincent Noireaux4 1Department of Biology, California Institute of Technology, 2Department of Bioengineering, California Institute of Technology, 3Synthetic Biology Center, Department of Bioengineering, Massachusetts Institute of Technology, 4School of Physics and Astronomy, University of Minnesota This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data. Engineering Solid-state Graft Copolymer Electrolytes for Lithium Battery Applications Qichao Hu1, Antonio Caputo2, Donald R. Sadoway1 1Materials Science and Engineering, Massachusetts Institute of Technology, 2Materials Processing Center, Massachusetts Institute of Technology Lithium ion batteries employ flammable and volatile organic electrolytes that are suitable for ambient temperature applications. A safer alternative to organic electrolytes are solid polymer batteries. Solid polymer batteries operate safely at high temperatures (>120 °C), thus making them applicable to high temperature applications such as deep oil drilling and hybrid electric vehicles. This paper will discuss (a) the polymer synthesis, (b) the polymer conduction mechanism, and (c) provide temperature cycling for both solid polymer and organic electrolytes. Engineering Scanning-probe Single-electron Capacitance Spectroscopy Kathleen A. Walsh1, Megan E. Romanowich1, Morewell Gasseller1,2, Irma Kuljanishvili1,3, Raymond Ashoori4, Stuart Tessmer1 1Department of Physics and Astronomy, Michigan State University, 2Department of Chemistry & Biochemistry/Physics, Mercyhurst University, 3Department of Physics, Saint Louis University, 4Department of Physics, Massachusetts Institute of Technology Scanning-probe single-electron capacitance spectroscopy facilitates the study of single-electron motion in localized subsurface regions. A sensitive charge-detection circuit is incorporated into a cryogenic scanning probe microscope to investigate small systems of dopant atoms beneath the surface of semiconductor samples. Biology Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS Christopher J. Zopf1, Narendra Maheshri1 1Department of Chemical Engineering, Massachusetts Institute of Technology We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data. 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. Biology Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans Elizabeth C. Pino1, Christopher M. Webster1, Christopher E. Carr2, Alexander A. Soukas1 1Center for Human Genetic Research and Department of Medicine, Massachusetts General Hospital and Harvard Medical School, 2Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry. Biology The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli Ming Jiang1, Haoran Zhang2, Blaine A. Pfeifer1 1Chemical and Biological Engineering Department, State University of New York at Buffalo, 2Chemical Engineering Department, Massachusetts Institute of Technology The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host. Neuroscience Manual Drainage of the Zebrafish Embryonic Brain Ventricles Jessica T. Chang1, Hazel Sive1 1Department of Biology, Whitehead Institute of Biomedical Research, Massachusetts Institute of Technology We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development. Engineering Micro 3D Printing Using a Digital Projector and its Application in the Study of Soft Materials Mechanics Howon Lee1, Nicholas X. Fang1 1Department of Mechanical Engineering, Massachusetts Institute of Technology We demonstrate controlled pattern transformation of swelling gel tubes by elastic instability. A simple projection micro stereo-lithography setup is built using an off-the-shelf digital data projector to fabricate three-dimensional polymeric structures in a layer-by-layer fashion. Swelling hydrogel tubes under mechanical constraint display various circumferential buckling modes depending on dimension. Neuroscience An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium Jessica T. Chang1,2, Hazel Sive1,2 1Department of Biology, Massachusetts Institute of Technology, 2Whitehead Institute of Biomedical Research We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease. Neuroscience Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat Elizabeth D. Kirby1, Kelly Jensen2, Ki A. Goosens3, Daniela Kaufer1,4 1Helen Wills Neuroscience Institute, University of California Berkeley, 2Office of Laboratory Animal Care, University of California Berkeley, 3McGovern Institute for Brain Research & The Department of Brain and Cognitive Science, Massachusetts Institute of Technology, 4Integrative Biology Department, University of California Berkeley Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain. Bioengineering Polymer Microarrays for High Throughput Discovery of Biomaterials Andrew L. Hook1, Chien-Yi Chang2, Jing Yang1, David J. Scurr1, Robert Langer3, Daniel G. Anderson3, Steve Atkinson2, Paul Williams2, Martyn C. Davies1, Morgan R. Alexander1 1Laboratory of Biophysics and Surface Analysis, University of Nottingham, 2School of Molecular Medical Sciences, University of Nottingham, 3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described. Immunology and Infection Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging Joel R. Meyerson1,2, Tommi A. White1, Donald Bliss3, Amy Moran3, Alberto Bartesaghi1, Mario J. Borgnia1, M. Jason V. de la Cruz1, David Schauder1, Lisa M. Hartnell1, Rachna Nandwani1,4, Moez Dawood5, Brianna Kim6, Jun Hong Kim7, John Sununu8, Lisa Yang9, Siddhant Bhatia10, Carolyn Subramaniam1, Darrell E. Hurt11, Laurent Gaudreault12, Sriram Subramaniam1 1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia, 7Duke University, 8Yale University, 9University of Notre Dame, 10Washington University in St. Louis, 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis. Biology Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry Lei Zhao*1, Jeffrey R. Whiteaker*1, Matthew E. Pope2, Eric Kuhn3, Angela Jackson4, N. Leigh Anderson5, Terry W. Pearson2, Steven A. Carr3, Amanda G. Paulovich1 1Clinical Research Division, Fred Hutchinson Cancer Research Center - FHCRC, 2Department of Biochemistry and Microbiology, University of Victoria, 3Broad Institute of MIT and Harvard, 4Genome BC Proteomics Centre, University of Victoria, 5Plasma Proteome Institute Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format. Bioengineering Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns Chia-Hua Lee1, Suman Bose2, Krystyn J. Van Vliet1, Jeffrey M. Karp3, Rohit Karnik2 1Department of Materials Science and Engineering, MIT - Massachusetts Institute of Technology, 2Department of Mechanical Engineering, MIT - Massachusetts Institute of Technology, 3HST Center for Biomedical Engineering and Harvard Stem Cell Institute, Brigham and Women's Hospital and Harvard Medical School We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis. Immunology and Infection Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT Oktay Kirak1, Eva-Maria Frickel1, Gijsbert M. Grotenbreg1,2, Heikyung Suh1, Rudolf Jaenisch1,3, Hidde L. Ploegh1,3 1Whitehead Institute for Biomedical Research, 2Departments of Microbiology and Biological Sciences, National University of Singapore, 3Department of Biology, Massachusetts Institute of Technology We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter. Biology Hi-C: A Method to Study the Three-dimensional Architecture of Genomes. Nynke L. van Berkum*1, Erez Lieberman-Aiden*2,3,4,5, Louise Williams*2, Maxim Imakaev6, Andreas Gnirke2, Leonid A. Mirny3,6, Job Dekker1, Eric S. Lander2,7,8 1Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Broad Institute of Harvard and Massachusetts Institute of Technology, 3Division of Health Sciences and Technology, Massachusetts Institute of Technology, 4Program for Evolutionary Dynamics, Department of Organismic and Evolutionary Biology, Department of Mathematics, Harvard University, 5Department of Applied Mathematics, Harvard University, 6Department of Physics, Massachusetts Institute of Technology, 7Department of Systems Biology, Harvard Medical School, 8Department of Biology, Massachusetts Institute of Technology The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale. Neuroscience Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits Stephanie Chan1, Jacob Bernstein1, Edward Boyden1 1Biological Engineering, Brain and Cognitive Sciences, and McGovern Institute, Massachusetts Institute of Technology Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits. Biology Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System Brad Hamilton1, Qiang Feng1, Mike Ye1, G Grant Welstead2 1Stemgent, 2Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers. Biology Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE) Heike Summer1, René Grämer2, Peter Dröge1 1School of Biological Sciences, Nanyang Technological University, Singapore - NTU, 2Singapore-MIT Alliance for Reserach and Technology (SMART) Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight. Biology Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly David P. Nguyen1,2, Stuart P. Layton1,2, Gregory Hale1,2, Stephen N. Gomperts1,2, Thomas J. Davidson1,2, Fabian Kloosterman1,2, Matthew A. Wilson1,2 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. Biology Micro-drive Array for Chronic in vivo Recording: Drive Fabrication Fabian Kloosterman1,2, Thomas J. Davidson1,2, Stephen N. Gomperts1,2, Stuart P. Layton1,2, Gregory Hale1,2, David P. Nguyen1,2, Matthew A. Wilson1,2 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. Biology Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 3 Judy Yen1, Ron Golan1, Kathleen Rubins1 1Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3. Biology Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2 Judy Yen1, Ron Golan1, Kathleen Rubins1 1Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3. Biology Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1 Judy Yen1, Ron Golan1, Kathleen Rubins1 1Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3. Biology Zebrafish Brain Ventricle Injection Jennifer H. Gutzman1, Hazel Sive1,2 1Whitehead Institute for Biochemical Research, 2MIT - Massachusetts Institute of Technology After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo. Biology Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy Ellie Graeden1,2, Hazel Sive1,2 1Department of Biology, MIT - Massachusetts Institute of Technology, 2Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain. Biology Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis Randal Halfmann1,2,3, Susan Lindquist1,2,3 1Whitehead Institute for Biomedical Research, 2Department of Biology, MIT - Massachusetts Institute of Technology, 3Howard Hughes Medical Institute SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications. Biology Interview: Protein Folding and Studies of Neurodegenerative Diseases Susan Lindquist1 1Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms. Biology Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4 G. Grant Welstead1, Tobias Brambrink1, Rudolf Jaenisch1 1Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4. Biology Patterning of Embryonic Stem Cells Using the Bio Flip Chip Nikhil Mittal1,2, Stephanie Flavin2, Joel Voldman2 1Dept of Physics, MIT - Massachusetts Institute of Technology, 2Department of Electrical Engineering and Computer Science, MIT - Massachusetts Institute of Technology We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells. Biology Micro-scale Engineering for Cell Biology Joel Voldman1 1Department of Electrical Engineering and Computer Science, MIT - Massachusetts Institute of Technology 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 Silicon Microchips for Manipulating Cell-cell Interaction Elliot E Hui1, Sangeeta N Bhatia1 1Laboratory for Multiscale Regenerative Technologies, MIT - Massachusetts Institute of Technology This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis. Biology Large-Scale Screens of Metagenomic Libraries Vinh D. Pham1, Tsultrim Palden1, Edward F. DeLong1 1Division of Biological Engineering, Department of Civil and Environmental Engineering, MIT - Massachusetts Institute of Technology Biology Microbial Communities in Nature and Laboratory - Interview Edward F. DeLong1 1Division of Biological Engineering, Department of Civil and Environmental Engineering, MIT - Massachusetts Institute of Technology Biology Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers Justin R. Seymour1, Marcos1, Roman Stocker1 1Environmental Microfluidics Group, MIT - Massachusetts Institute of Technology The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described. Biology Studies of Bacterial Chemotaxis Using Microfluidics - Interview Roman Stocker1 1Environmental Microfluidics Group, MIT - Massachusetts Institute of Technology
