Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.
1Department of Medical Oncology, Dana-Farber Cancer Institute, 2Harvard Medical School, Boston, MA, 3Sheba Cancer Research Center, Chaim Sheba Medical Center, 4Department of Pathology, Brigham and Women's Hospital
The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.
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.
A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.
Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis
1Division of Microbiology, Tulane National Primate Research Center, 2Division of Comparative Pathology, Tulane National Primate Research Center, 3Division of Veterinary Medicine, Tulane National Primate Research Center
Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.
Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice
1Department of Microbiology and Immunology, University of Melbourne, 2Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, 3The Centre for Dynamic Imaging, The Walter and Eliza Hall Institute for Medical Research
A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.
Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)
The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.
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.
Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.
Experimental rat endocarditis model due to methicillin-resistant S. aureus.
This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.
An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.
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.
We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.
A screening method to detect oxidative cellular environments is to measure the oxidation of CM-H2DCFDA. Once oxidized within a cell, CM-H2DCFDA changes from non-fluorescent into a fluorescent compound. This change in fluorescence is measured by flow cytometry and indicates the number of cells in an oxidative environment.
1Research Service, Veterans Administration Medical Center, Memphis, TN, 2Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, 3Department of Anatomy/Neurobiology, University of Tennessee Health Science Center, Memphis, TN
A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the December 2012 Issue of Journal of Visualized Experiments (JoVE).
Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression
We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.
The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis
Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.
One of the pathological characteristics of AD is the formation of Amyloid β protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.
1Defence Medical and Environmental Research Institute, DSO National Laboratories, 2Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School
Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.
Here are some highlights from the February 2012 Issue of Journal of Visualized Experiments (JoVE).
1Department of Pediatrics, University of Texas Southwestern Medical Center, 2Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 3Department of Pediatrics and Microbiology, University of Texas Southwestern Medical Center
A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.
The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.
Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples
Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary. This allowed for the detection of the bacterium using fluorescent microscopy.
Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1β using a Whole Blood Stimulation Assay
1Translational Immunology Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, 2Translational Autoinflammatory Disease Section, Office of the Clinical Director, National Institute of Arthritis and Musculoskeletal and Skin Diseases
We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.
C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.
Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV’s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.
Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.
Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.
The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.
Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.
We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.
We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.
1Department of Family and Community Medicine, Thomas Jefferson University, 2Department of Pharmacology and Experimental Therapeutics, Biostatistics Division, Thomas Jefferson University, 3Department of Internal Medicine, Thomas Jefferson University
Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.
1Department of Microbiology and Immunology, Tulane University Medical School, 2Physician/Scientist Program, Tulane University Medical School, 3Department of Molecular and Cellular Biology, Baylor College of Medicine
Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.
Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.
A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.
The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.
Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.
An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.
SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval
1Yerkes National Primate Research Center, Emory University, 2Department of Neurology, Institute of Clinical Medicine, Tsukuba University, 3Department of Pathology, New York University School of Medicine, 4Department of Neurology, Emory University
We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the Aβ peptide. Using this protocol, we consistently detect monomeric and multimeric Aβ in cortical tissue from humans with Alzheimer's pathology.
Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.
We report a method to isolate naïve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.
Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.
A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.
Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.
Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood
We describe use of ImageStream technology (www.amnis.com), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.
A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.
The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.