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HIV-1: The type species of Lentivirus and the etiologic agent of Aids. It is characterized by its cytopathic effect and affinity for the T4-lymphocyte.
 JoVE Immunology and Infection

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

1Emory Vaccine Center at Yerkes National Primate Research Center, Emory University, 2Department of Pathology and Laboratory Medicine, Emory University

JoVE 51506

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

 JoVE Medicine

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

1Africa Centre for Health and Population Studies, College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa, 2Unit D11, Jembi Health Systems, 3Academic Medical Center, Department of Global Health, Amsterdam Institute for Global Health and Development (AIGHD), University of Amsterdam, 4Division of Infectious Diseases and Geographic Medicine, Centre for AIDS Research, Stanford Medical School

JoVE 51242

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

 JoVE Immunology and Infection

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

1Department of Immunology and Microbiology, Rush University Medical Center

JoVE 2668

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 JoVE Immunology and Infection

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

1The virology Core at the HIV Drug Resistance Program, NCI-Frederick, 2Division of Infectious Diseases, University of Pittsburgh, 3Department of Molecular Biology and Microbiology, Tuffts University

JoVE 2960

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

 JoVE Immunology and Infection

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

1Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 2The RNA Institute, SUNY Albany

JoVE 52474

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

 JoVE Immunology and Infection

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

1Ragon Institute of MGH, MIT, and Harvard, 2Division of Infectious Diseases, Massachusetts General Hospital

JoVE 50244

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

 JoVE Immunology and Infection

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

1Infectious Disease Research Center, CHUL (CHUQ), Quebec City, Quebec, Canada

JoVE 4166

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

 JoVE Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

1Laboratory Program, BC Centre for Excellence in HIV/AIDS

JoVE 2531

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

 JoVE Immunology and Infection

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

1Institute of Virology, University of Cologne, 2Max Planck Institute for Informatics, 3Institute for Immune genetics, 4Department of Gastroenterology, Hepatology and Infectiology, University of Duesseldorf, 5Department of Dermatology, University of Essen, 6Department of Internal Medicine, University of Cologne, 7Augustinerinnen Hospital

JoVE 3264

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

 JoVE Immunology and Infection

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

1Centre de recherche du CHUM, Department of Microbiology, Infectiology and Immunology, Université de Montréal

JoVE 51995

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

 JoVE Immunology and Infection

In vitro Uncoating of HIV-1 Cores

1Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine

JoVE 3384

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

 JoVE Immunology and Infection

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

1National Center for Biodefense and Infectious Diseases, Department of Molecular and Microbiology, George Mason University

JoVE 2198

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

 JoVE Immunology and Infection

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

1Department of Pathology, New York University Langone School of Medicine, 2Program in Molecular Pathogenesis, Marty and Helen Kimmel Center for Biology and Medicine and Skirball Institute for Biomolecular Medicine, 3Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 4Veteran Affairs New York Harbor Healthcare System

JoVE 3757

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

 JoVE Immunology and Infection

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

1Department of Structural Biology, University of Pittsburgh School of Medicine

JoVE 3041

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

 JoVE Immunology and Infection

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

1Department of Pharmacology, School of Medicine, New York University

JoVE 2118

The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.

 JoVE Biology

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

1Department of Cell Biology and Virology, Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg

JoVE 793

Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.

 JoVE Biology

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

1Department of Bioengineering, Rice University

JoVE 52620

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

 JoVE Immunology and Infection

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

1Department of Microbiology, University of Washington, 2Departments of Medicine and Laboratory Medicine, University of Washington, 3U.S Military HIV Research Program, Walter Reed Army Institute of Research, 4Henry M. Jackson Foundation

JoVE 52610

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

 JoVE Biology

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

1Department of Chemical and Biological Engineering, Princeton University

JoVE 50476

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design ( Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

 JoVE Immunology and Infection

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

1Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, 2Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 3Shared Resource-DNA/RNA Peptide, Beckman Research Institute of City of Hope

JoVE 2954

Several 2’-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

 JoVE Immunology and Infection

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

1Department of Immunology, John Curtin School of Medical Research, Australian National University

JoVE 51627

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses >250 parameters simultaneously by flow cytometry.

 JoVE Medicine

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

1Department of Medical Microbiology, University of Manitoba, 2Department of Community Health Sciences, University of Manitoba

JoVE 51906

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

 JoVE Immunology and Infection

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

1The Ragon Institute of MGH, MIT and Harvard, 2Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM)

JoVE 50821

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

 JoVE Immunology and Infection

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

1HIV Drug Resistance Program, National Cancer Institute

JoVE 51400

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

 JoVE Biology

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

1Institute of Virology, National Reference Centre for Hepatitis C, Essen University Hospital, University of Duisburg-Essen

JoVE 52619

The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.

 JoVE Medicine

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty

1Division of Infectious Diseases, University of Arizona, 2Arizona Center on Aging, University of Arizona

JoVE 50537

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

 JoVE Immunology and Infection

Peptide-based Identification of Functional Motifs and their Binding Partners

1Department of Microbiology, Biochemistry, & Immunology, Morehouse School of Medicine, 2Institute for Systems Biology, 3Advanced Medical & Dental Institute, Universiti Sains Malaysia

JoVE 50362

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef’s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

 JoVE Immunology and Infection

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

1Department of Medicine, University of Alabama at Birmingham, 2Center for AIDS Research, University of Alabama at Birmingham

JoVE 52655

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

 JoVE Bioengineering

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

1Department of Structural Biology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine

JoVE 50386

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

 JoVE Immunology and Infection

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

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

JoVE 2770

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.

 JoVE Bioengineering

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

1Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center

JoVE 2460

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.

 JoVE Medicine

The α-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

1Department of Medicine, Weill Cornell Medical College, 2Department of Oral Biology, University of Missouri-Kansas City-School of Dentistry, 3Department of Pharmacology and Toxicology, University of Missouri Kansas City- School of Pharmacy, 4Regional Hospital, Bamenda, NWP, Cameroon, 5Mezam Polyclinic HIV/AIDS Treatment Center, Cameroon, 6Institute for Human Genetics and Biochemistry

JoVE 3999

A CD4 enumeration method, the α-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The α-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

 JoVE Biology

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 2Department of Microbiology and Immunology, McGill University, 3Department of Medicine, Division of Experimental Medicine, McGill University

JoVE 4002

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

 JoVE Immunology and Infection

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

1Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, 2NSF Center for Biophotonics, University of California, Davis, 3Structural and Computational Biology Unit, European Molecular Biology Laboratory

JoVE 2061

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

 JoVE Biology

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

1Department of Biology, San Diego State University

JoVE 52452

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

 JoVE Biology

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

1Department of Infectious Diseases, The Scripps Research Institute, 2Department of Chemistry, City College of New York

JoVE 51309

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

 JoVE Biology

RNA Secondary Structure Prediction Using High-throughput SHAPE

1RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research

JoVE 50243

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

 JoVE Immunology and Infection

Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

1Unité d'Immunopathologie Virale, Centre de Recherche du CHU Sainte-Justine, Department of Microbiology, Infectiology & Immunology, Faculty of Medicine, Université de Montréal, 2Infectious Diseases Service, CHU Sainte-Justine, Faculty of Medicine, Université de Montréal, 3Department of Paediatrics, Université de Montréal

JoVE 51643

Novel generations of functional assays such as gamma interferon (IFN-γ) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

 JoVE Neuroscience

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

1Department of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine

JoVE 4031

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

 JoVE Immunology and Infection

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

1Department of Medicine, Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, 2UCLA AIDS Institute, 3Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, 4Department of Medical and Molecular Pharmacology, David Geffen School of Medicine at UCLA, 5Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA

JoVE 4181

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

 JoVE Neuroscience

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

1Center for Neural Development and Disease, Department of Neurology, Child Neurology Division, University of Rochester, 2Department of Neurobiology and Anatomy, University of Rochester

JoVE 2059

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.

 JoVE Immunology and Infection

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

1Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles

JoVE 50606

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

 JoVE Immunology and Infection

Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

1Department of Microbiology and Parasitology, College of Basic Medical Sciences, China Medical University, 2Department of Obstetrics and Gynecology, College of Medicine, University of Arkansas for Medical Sciences, 3Department of Pathology, College of Medicine, University of Arkansas for Medical Sciences

JoVE 3657

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

 JoVE Immunology and Infection

Determining the Phagocytic Activity of Clinical Antibody Samples

1Massachusetts General Hospital, Ragon Institute of MGH, MIT, and Harvard, 2Thayer School of Engineering, Dartmouth College

JoVE 3588

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors—providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

 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

JoVE 2459

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 Biology

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

1DNA Medicine Institute, 2Harvard Medical School, 3NASA Glenn Research Center, 4ZIN Technologies

JoVE 51743

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

 JoVE Biology

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

1Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology, 2Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine

JoVE 51666

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

 JoVE Immunology and Infection

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

1CREA, Diagnostics Department, Spedali Civili di Brescia

JoVE 52184

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

 JoVE Immunology and Infection

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

1Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford

JoVE 51714

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

 JoVE Neuroscience

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

1College of Veterinary Medicine, Western University of Health Sciences, 2Graduate College of Biomedical Sciences, Western University of Health Sciences, 3ReadiSorb, Products

JoVE 2841

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.

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