University of Rochester View Institution's Website 46 articles published in JoVE Bioengineering Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems Mehran Mansouri1, Aidan R. Hughes1, Lauren A. Audi1, Anna E. Carter1, Justin A. Vidas1, James L. McGrath2, Vinay V. Abhyankar1 1Department of Biomedical Engineering, Rochester Institute of Technology, 2Department of Biomedical Engineering, University of Rochester This protocol describes a reconfigurable membrane-based cell culture platform that integrates the open-well format with fluid flow capabilities. This platform is compatible with standard protocols and allows for reversible transitions between open-well and microfluidic culture modes, accommodating the needs of both engineering and bioscience laboratories. Bioengineering Use of the MicroSiM (µSiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier Molly C. McCloskey1, Pelin Kasap2, Michelle Trempel1, Louis P. Widom3, Julia Kuebel1, Kaihua Chen1, Thomas R. Gaborski3, Britta Engelhardt2, James L. McGrath1 1Department of Biomedical Engineering, University of Rochester, 2Theodor Kocher Institute, University of Bern, 3Department of Biomedical Engineering, Rochester Institute of Technology This report provides protocols for assembly, cell culture, and assays on the µSiM platform for the construction of blood-brain barrier models. Bioengineering Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement Brian C. Wise1,2, Keshia E. Mora1,2, Whasil Lee1,2,3, Mark R. Buckley1,2 1Department of Biomedical Engineering, University of Rochester, 2Department of Orthopaedics, Center for Musculoskeletal Research, University of Rochester Medical Center, 3Department of Pharmacology and Physiology, University of Rochester Medical Center We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement. Cancer Research Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia Christina M. Kaszuba1,2, Benjamin J. Rodems1,3, Sonali Sharma1,3, Edgardo I. Franco1,2, John M. Ashton1,3,4, Laura M. Calvi1,5, Jeevisha Bajaj1,3 1Wilmot Cancer Institute, University of Rochester Medical Center, 2Department of Biomedical Engineering, University of Rochester, 3Department of Biomedical Genetics, University of Rochester Medical Center, 4Genomics Research Center, University of Rochester Medical Center, 5Division of Endocrinology and Metabolism, Department of Medicine, University of Rochester Medical Center Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression. Neuroscience Electrophysiology of Laminar Cortical Activity in the Common Marmoset Amy Bucklaew1, Shanna H. Coop2, Jude F. Mitchell1,2 1Neuroscience, University of Rochester, 2Brain and Cognitive Sciences, University of Rochester Custom-built micro-drives enable the sub-millimeter targeting of cortical recording sites with linear silicon arrays. Bioengineering Cantilever Bending of Murine Femoral Necks Emma Knapp1, Hani A. Awad1,2 1Department of Orthopedics, Center for Musculoskeletal Research, University of Rochester Medical Center, 2Department of Biomedical Engineering, University of Rochester The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment. Developmental Biology Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos Marcus D. Kilwein1, Michael A. Welte1 1Department of Biology, University of Rochester In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry. Immunology and Infection A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection Kevin Chiem1, Javier Rangel-Moreno2, Aitor Nogales1,3, Luis Martinez-Sobrido1 1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Division of Allergy/Immunology and Rheumatology, Department of Medicine, University of Rochester, 3Center for Animal Health Research, INIA-CISA Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo. Bioengineering Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter Ewa M. Spiesz*1, Kui Yu*1, Benjamin A.E. Lehner1, Dominik T. Schmieden1, Marie-Eve Aubin-Tam1, Anne S. Meyer2 1Department of Bionanoscience & Kavli Institute of Nanoscience, Delft University of Technology, 2Department of Biology, University of Rochester This article describes a method of transforming a low-cost commercial 3D printer into a bacterial 3D printer that can facilitate printing of patterned biofilms. All necessary aspects of preparing the bioprinter and bio-ink are described, as well as verification methods to assess the formation of biofilms. Bioengineering Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants Alexander Kotelsky1, Joseph S. Carrier1, Mark R. Buckley1 1Department of Biomedical Engineering, University of Rochester We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes. Medicine Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation Jomy J. Varghese1, Isaac L. Schmale2, Mollie Eva Hansen1, Shawn D. Newlands2, Danielle S.W. Benoit1, Catherine E. Ovitt3 1Department of Biomedical Engineering, University of Rochester, 2Department of Otolaryngology, University of Rochester Medical Center, 3Center for Oral Biology, University of Rochester Medical Center We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands. Bioengineering Retroductal Nanoparticle Injection to the Murine Submandibular Gland Jomy J. Varghese1, Isaac L. Schmale2, Yuchen Wang1, Mollie Eva Hansen1, Shawn D. Newlands2, Catherine E. Ovitt3, Danielle S. W. Benoit1 1Department of Biomedical Engineering, University of Rochester, 2Department of Otolaryngology Head and Neck Surgery, University of Rochester Medical Center, 3Center for Oral Biology, University of Rochester Medical Center Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles. Biochemistry Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution Gisela Beutner1, George Arthur Porter Jr.1 1Department of Pediatrics-Division Cardiology, University of Rochester This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes. Biology High-Throughput, Multi-Image Cryohistology of Mineralized Tissues Nathaniel A. Dyment1, Xi Jiang1, Li Chen1, Seung-Hyun Hong2, Douglas J. Adams3, Cheryl Ackert-Bicknell4, Dong-Guk Shin2, David W. Rowe1 1Department of Reconstructive Sciences, University of Connecticut Health Center, 2Department of Computer Science and Engineering, University of Connecticut, 3Department of Orthopaedic Surgery, University of Connecticut Health Center, 4Department of Orthopaedics, University of Rochester In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues. Immunology and Infection Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis Alison Gaylo1, Michael G. Overstreet1, Deborah J. Fowell1 1David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ. Engineering Ultrasound Velocity Measurement in a Liquid Metal Electrode Adalberto Perez1, Douglas H. Kelley1 1Department of Mechanical Engineering, University of Rochester Ultrasound velocimetry is used to study mixing by fluid flow in liquid metal electrodes. The focus of this manuscript is to illustrate the methods used for making precise, spatially-resolved ultrasound measurements while limiting oxidation and controlling and monitoring temperature, applied current, and the heater power being supplied. Behavior Methods to Test Visual Attention Online Amanda Yung1, Pedro Cardoso-Leite2, Gillian Dale3, Daphne Bavelier2,4, C. Shawn Green3 1Center for Visual Science, University of Rochester, 2Faculty of Psychology and Educational Sciences, University of Geneva, 3Department of Psychology, University of Wisconsin-Madison, 4Department of Brain and Cognitive Sciences, University of Rochester To replicate laboratory settings, online data collection methods for visual tasks require tight control over stimulus presentation. We outline methods for the use of a web application to collect performance data on two tests of visual attention. Biology Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes Amanda M. Larracuente1, Patrick M. Ferree2 1Department of Biology, University of Rochester, 2W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals. Biology A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity Matthew D. Rand1 1Department of Environmental Medicine, University of Rochester School of Dentistry and Medicine Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model. Chemistry Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation Amy H. Van Hove1, Brandon D. Wilson2, Danielle S. W. Benoit1,2,3 1Department of Biomedical Engineering, University of Rochester, 2Department of Chemical Engineering, University of Rochester, 3Center for Musculoskeletal Research, University of Rochester Medical Center This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques. Immunology and Infection Rescue of Recombinant Newcastle Disease Virus from cDNA Juan Ayllon1,2, Adolfo García-Sastre1,2,3, Luis Martínez-Sobrido4 1Department of Microbiology, Icahn School of Medicine at Mount Sinai, 2Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, 3Department of Medicine, Icahn School of Medicine at Mount Sinai, 4Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here. Medicine Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia Tristan M. Nicholson1,2, Kristen S. Uchtmann1, Conrad D. Valdez1, Ashleigh B. Theberge1,3, Tihomir Miralem1, William A. Ricke1 1Department of Urology, University of Wisconsin-Madison, 2Medical Scientist (MD/PhD) Training Program, University of Rochester School of Medicine & Dentistry, 3Molecular and Environmental Toxicology Center, University of Wisconsin-Madison We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth. Immunology and Infection Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells Benson Y.H. Cheng*1, Emilio Ortiz-Riaño*1, Juan Carlos de la Torre2, Luis Martínez-Sobrido1 1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Immunology and Microbial Sciences, The Scripps Research Institute Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses. Neuroscience Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining Maria Cecilia Barone1, Dirk Bohmann1 1Department of Biomolecular Genetics, University of Rochester Medical Center Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts. Bioengineering Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy Graham Marsh1, Richard E. Waugh1 1Department of Biomedical Engineering, University of Rochester The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus. Medicine A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis Michele M. Pelter1, Teri M. Kozik2, Denise L. Loranger1, Mary G. Carey3 1Orvis School of Nursing, University of Nevada, Reno, 2 Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia. Neuroscience Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence Oksana Polesskaya1, Anita Sun2, Gheorghe Salahura2, Jharon N. Silva1, Stephen Dewhurst1, Karl Kasischke3 1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Center for Neural Development and Disease, University of Rochester Medical Center, 3Deptartment of Neurology, Center for Neural Development and Disease, University of Rochester Medical Center Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply. Bioengineering Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium Carleen M. Bowers1, Eric J. Toone1, Robert L. Clark2, Alexander A. Shestopalov3 1Department of Chemistry, Duke University, 2Hajim School of Engineering and Applied Sciences, University of Rochester, 3Department of Chemical Engineering, University of Rochester Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns. Medicine The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse Gozde Colak1, Anthony J. Filiano2, Gail V.W. Johnson3 1Department of Pharmacology and Physiology, University of Rochester, 2Department of Neurology, University of Alabama at Birmingham, 3Departments of Anesthesiology, Pharmacology and Physiology, University of Rochester Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available. Immunology and Infection An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci Marlise I. Klein1, Jin Xiao1,2, Arne Heydorn3, Hyun Koo1,4 1Center for Oral Biology, University of Rochester Medical Center, 2State Key Laboratory of Oral Diseases, Sichuan University, 3Department of General Medicine, Glostrup Hospital, Glostrup, Denmark, 4Department of Microbiology and Immunology, University of Rochester Medical Center Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research. Neuroscience Intracranial Injection of Adeno-associated Viral Vectors Rebecca L. Lowery1, Ania K. Majewska1 1Neurobiology and Anatomy, University of Rochester Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex. Neuroscience Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation Emily A. Kelly1, Ania K. Majewska1 1Neurobiology and Anatomy, University of Rochester In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. Biology Establishing Primary Adult Fibroblast Cultures From Rodents Andrei Seluanov1, Amita Vaidya1, Vera Gorbunova1 1Department of Biology, University of Rochester This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents. Neuroscience A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation Daniel F. Marker*1, Marie-Eve Tremblay*2, Shao-Ming Lu1, Ania K. Majewska2, Harris A. Gelbard1 1Center for Neural Development and Disease, Department of Neurology, Child Neurology Division, University of Rochester, 2Department of Neurobiology and Anatomy, University of Rochester We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat. Immunology and Infection Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis Hristina Nedelkovska1, Tanya Cruz-Luna1, Pamela McPherson1, Jacques Robert1 1Department of Microbiology and Immunology, University of Rochester The frog Xenopus laevis provides an attractive alternative non-mammalian model for exploring the ability of heat shock protein such as gp96 to promote antigen-specific CD8 T cell responses. We present methods to study in vivo facilitation of cross-presentation of skin and tumor antigens by gp96. Biology Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells Andrei Seluanov1, Zhiyong Mao1, Vera Gorbunova1 1Department of Biology, University of Rochester This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells. Medicine Experimental Metastasis Assay Sonali Mohanty1, Lei Xu1,2 1Department of Biomedical Genetics, University of Rochester Medical Center, 2Department of Dermatology, University of Rochester Medical Center This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines. Neuroscience Double Fluorescence in situ Hybridization in Fresh Brain Sections Jin Kwon Jeong1, Zhuoxun Chen1, Liisa A. Tremere1, Raphael Pinaud1,2 1Department of Brain and Cognitive Sciences, University of Rochester, 2Center for Visual Science, University of Rochester This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain. Medicine Murine Echocardiography and Ultrasound Imaging Andrew Pistner1, Stephen Belmonte2, Tonya Coulthard3, Burns C. Blaxall2,4 1Department of Pharmacology and Physiology, University of Rochester, 2Aab Cardiovascular Research Institute, University of Rochester, 3Visualsonics, 4Department of Medicine, University of Rochester This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging. Immunology and Infection Generation of Recombinant Influenza Virus from Plasmid DNA Luis Martínez-Sobrido1, Adolfo García-Sastre2 1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Microbiology and Medicine, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines. Neuroscience Preparation of Mouse Brain Tissue for Immunoelectron Microscopy Marie-Eve Tremblay1, Mustapha Riad2, Ania Majewska1 1Department of Neurobiology and Anatomy, University of Rochester, 2Douglas Mental Health University Institute We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy. Biology In-vivo Centrifugation of Drosophila Embryos Susan L. Tran1, Michael A. Welte1 1Department of Biology, University of Rochester We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments. Biology Derivation of Mouse Trophoblast Stem Cells from Blastocysts Shang-Yi Chiu1, Eri O. Maruyama1, Wei Hsu1 1Department of Biomedical Genetics, Center for Oral Biology, James P Wilmot Cancer Center, University of Rochester In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture. Immunology and Infection Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro Craig T. Lefort1, Minsoo Kim1 1Center for Vaccine Biology and Immunology, University of Rochester T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro. Biology Chronic Salmonella Infected Mouse Model Shaoping Wu*1, Rong Lu*1, Yong-guo Zhang1, Jun Sun1 1Department of Medicine, University of Rochester Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks. Biology Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching Kelley D. Sullivan1, Edward B. Brown2 1Department of Physics and Astronomy, University of Rochester, 2Department of Biomedical Engineering, University of Rochester In this article we will describe the procedure for measuring diffusion coefficients using multi-photon fluorescence recovery after photobleaching. We will begin by aligning the laser along the optical path to the sample and determining the proper experimental parameters, then continue generating and finally fitting fluorescence recovery curves.
