Lymph Nodes:
They are oval or bean shaped bodies (1 - 30 mm in diameter) located along the lymphatic system.
JoVE General
Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)
Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.
JoVE Clinical and Translational Medicine
Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology
1Department of Surgery, Division of Surgical Oncology, University Medical Center Groningen, 2Helmholtz Zentrum, Technical University Munich, 3Department of Obstetrics and Gynaecology, University Medical Center Groningen
Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.
JoVE Immunology and Infection
Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes
1Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), 2Inserm, U1016, Paris, France
This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.
JoVE Immunology and Infection
Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging
1Experimental and Clinical Research Center, A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine, 2Berlin Ultrahigh Field Facility (B.U.F.F.), Max Delbrück Center for Molecular Medicine
Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.
JoVE Immunology and Infection
Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis
Louisiana State University Health Sciences Center
Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.
JoVE Immunology and Infection
Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge
1Department of Medicine, Section of Cardiology, St. John-Providence Health System, 2Department of Immunology and Microbiology, Wayne State University School of Medicine
Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.
JoVE Immunology and Infection
Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection
Department of Microbiology, Immunology and Pathology, Colorado State University
Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.
JoVE Immunology and Infection
Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells
Department of Microbiology & Immunology, Pennsylvania State University College of Medicine
We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.
JoVE Immunology and Infection
Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice
1Department of Environmental Medicine, New York University School of Medicine, Tuxedo, 2Division of Allergy, Pulmonary, & Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, 3Division of Pulmonary Medicine, New York University School of Medicine
A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.
JoVE Immunology and Infection
Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry
1Department of Immunology, University of Colorado School of Medicine, 2Division of Cell Biology, Department of Pediatrics, National Jewish Health, 3Department of Microbiology, Immunology, and Pathology, Colorado State University, 4Department of Immunology, National Jewish Health
We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.
JoVE Immunology and Infection
Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation
1Stem Cell and Cancer Research Institute, McMaster University, Hamilton, 2Physiology and Experimental Medicine Research Program, Hospital for Sick Children, University of Toronto
We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.
JoVE Immunology and Infection
Murine Superficial Lymph Node Surgery
1Maisonneuve-Rosemont Hospital Research Center, 2Department of Microbiology and Immunology, University of Montreal, 3Department of Medicine, University of Montreal
To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.
JoVE Clinical and Translational Medicine
Collection Protocol for Human Pancreas
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida
This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.
JoVE Immunology and Infection
Intravital Imaging of the Mouse Popliteal Lymph Node
1Department of Pediatrics, Case Western Reserve University, 2Department of Pediatrics, Pathology and Biomedical Engineering, Case Western Reserve University
Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.
JoVE Immunology and Infection
Intravital Microscopy of the Inguinal Lymph Node
1Interdisciplinary Science, University of Northern British Columbia, 2Northern Medical Program, University of Northern British Columbia
A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.
JoVE General
Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification
Department of Physiology and Biophysics, University of California, Irvine (UCI)
Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically ≥ 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.
JoVE Immunology and Infection
Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation
1Institute of Immunology, Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Pathobiology, School of Veterinary Medicine, University of Pennsylvania
Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.
JoVE Immunology and Infection
The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation
Department of Immunology, John Curtin School of Medical Research, Australian National University
CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.
JoVE Immunology and Infection
Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells
This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.
JoVE General
Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)
Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.
JoVE Immunology and Infection
Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy
1Institute for Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, 2Department of Immunoregulation, Helmholtz Center for Infection Research
We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).
JoVE Immunology and Infection
Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy
Laboratory of Immune Regulation, Instituto Gulbenkian de Ciência
We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of “niches” within the thymus where T cell development occurs.
JoVE Immunology and Infection
Competitive Homing Assays to Study Gut-tropic T Cell Migration
Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School
Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.
JoVE Immunology and Infection
Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse
Center for Molecular Imaging (CMI), University of Texas Health Science Center-Houston
Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.
JoVE General
Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice
1Research Animal Diagnostic Services, Charles River, 2Research Models and Services, Charles River, 3Department of Comparative Medicine, University of Washington
This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.
JoVE General
Isolation of Immune Cells from Primary Tumors
1Tumor Immunity and Tolerance Section, Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, National Cancer Institute - Frederick, 2KEWB Productions
In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.
JoVE General
Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats
1Department of Oncology, Georgetown University, 2Institute of Biomedicine, University of Turku Medical Faculty
Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.
JoVE Editorial
JoVE 7th Issue
JoVE Immunology and Infection
Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation
1Department of Microbiology & Immunology, Virginia Commonwealth University- Massey Cancer Center, 2Department of Internal Medicine, Virginia Commonwealth University- Massey Cancer Center, 3Department of Surgery, Virginia Commonwealth University- Massey Cancer Center
An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.
JoVE Immunology and Infection
Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky
This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.
JoVE Immunology and Infection
Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes
Department of Microbiology and Immunology, Pennsylvania State University College of Medicine
Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.
JoVE Immunology and Infection
Flow Cytometry Analysis of Immune Cells Within Murine Aortas
1Deptartment of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, 2Division of Inflammation Biology, LaJolla Institute for Allergy and Immunology
This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.
JoVE Clinical and Translational Medicine
Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer
1Department of Oncology, Georgetown University, 2Lombardi Comprehensive Cancer Center, Georgetown University, 3Stem Cell Dynamics, Helmholtz Zentrum München - German Research Center for Environmental Health, 4Department of Medicine, Georgetown University, 5Department of Nanobiomedical Science and WCU Research Center of Nanobiomedical Science, Dankook University
Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.
JoVE Immunology and Infection
Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow
1Department of poverty related diseases, Barcelona Centre for International Health Research, 2Confocal Microscopy Unit, University of Barcelona- Scientific and Technological Centers, 3Institució Catalana de Recerca i Estudis Avançats (ICREA)
We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.
JoVE Immunology and Infection
Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches
Division of Infectious Diseases, New York State Department of Health
There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.
JoVE Immunology and Infection
Isolation of Mouse Lung Dendritic Cells
Pathobiological Sciences, Louisiana State University
A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.
JoVE Immunology and Infection
Protein Misfolding Cyclic Amplification of Prions
1Department of Civil Engineering, University of Nebraska at Lincoln, 2Department of Medical Microbiology and Immunology, Creighton University
Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.
JoVE General
Dendra2 Photoswitching through the Mammary Imaging Window
1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine - Yeshiva University, 2Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine - Yeshiva University, 3Hubrecht Institute-KNAW and University Medical Center Utrecht
Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.
JoVE Immunology and Infection
Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines
U.S. Army Medical Research Institute of Infectious Diseases
Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.
JoVE Clinical and Translational Medicine
Intranasal Administration of CNS Therapeutics to Awake Mice
A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.
JoVE Clinical and Translational Medicine
Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma
1Institute of Pathology, Laboratory of Molecular Tumor Pathology, Charité - Universitätsmedizin Berlin, 2Institute for Chemistry and Biochemistry, Free University Berlin, 3Laboratory for Functional Genomics Charité (LFGC), Charité - Universitätsmedizin Berlin, 4Comprehensive Cancer Center Charité, Charité - Universitätsmedizin Berlin
This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).
JoVE Clinical and Translational Medicine
Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time
1Diabetes Research Institute, University of Miami Miller School of Medicine, 2Department of Surgery, University of Miami Miller School of Medicine, 3Department of Medicine, University of Miami Miller School of Medicine, 4Department of Physiology & Biophysics, University of Miami Miller School of Medicine, 5The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet
A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.
JoVE Immunology and Infection
Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut
1The Biomedical Research Centre, University of British Columbia, 2Department of Pathology and Laboratory Medicine, University of British Columbia
Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.
JoVE General
Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea
Department of Pediatrics, David Geffen School of Medicine at UCLA
Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.
JoVE Bioengineering
Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles
1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Veterinary Microbiology and Preventive Medicine, Iowa State University
Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.
JoVE Bioengineering
Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring
1Department of Radiology, University of Nebraska Medical Center, 2Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center
Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.
JoVE Clinical and Translational Medicine
An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth
1Department of Biology, University of Haifa, 2Transgenic Oncogenesis and Genomics Section, Laboratory of Cancer Biology and Genetics, National Cancer Institute
A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.
JoVE Immunology and Infection
Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies
1Molecular and Cell Biology Program, Ohio University, 2Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, 3Department of Biomedical Engineering, Russ College of Engineering and Technology, Ohio University
Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.
JoVE Clinical and Translational Medicine
Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)
Department of Molecular Medicine, University of South Florida Morsani College of Medicine
This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.
JoVE Bioengineering
Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs
Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center
Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.
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