Compensation and Redress:
Payment, or other means of making amends, for a wrong or injury.
JoVE Immunology and Infection
Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)
Blood Research Institute, BloodCenter of Wisconsin
For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.
JoVE Neuroscience
An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice
Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine
An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.
JoVE Immunology and Infection
Isolation of Adipose Tissue Immune Cells
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine
Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.
JoVE General
Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes
1Department of Flow and Image Cytometry, Roswell Park Cancer Institute, 2Flow Cytometry & Cell Sorting Resource Laboratory, University of Pennsylvania, 3SciGro, Inc., 4Department of Pathology and Laboratory Medicine, University of Pennsylvania
Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.
JoVE Bioengineering
Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry
1Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, 2Amnis Corporation, 3Department of Chemical and Biological Engineering, Iowa State University
In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.
JoVE Neuroscience
Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry
Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine
Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of β-galactosidase prior to separation via flow cytometry.
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 Immunology and Infection
Examination of Thymic Positive and Negative Selection by Flow Cytometry
Department of Medical Microbiology and Immunology, University of Alberta
We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.
JoVE Immunology and Infection
Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis
1Department of Kinesiology, Recreation, and Sport, Western Kentucky University, 2Department of Health and Human Performance, University of Houston
Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.
JoVE Immunology and Infection
Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky
We describe a method for generating regulatory, memory and naïve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.
JoVE Immunology and Infection
Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques
1Department of Immunology, MD Anderson Cancer Center - University of Texas, 2Department of Medicine, University of Miami
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.
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 Immunology and Infection
Isolation of Precursor B-cell Subsets from Umbilical Cord Blood
1Department of Pathology and Anatomical Sciences, University of Missouri-Columbia, 2Laboratory for Infectious Disease Research, University of Missouri-Columbia
Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.
JoVE General
Chromatin Isolation by RNA Purification (ChIRP)
ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.
JoVE Immunology and Infection
Induction and Assessment of Class Switch Recombination in Purified Murine B Cells
Department of Immunology, University of Toronto
Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.
JoVE General
Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging
1Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología-Universidad Nacional Autónoma de México, 2Math and Sciences Department, Edison State College
Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.
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 General
Fluorescence Activated Cell Sorting of Plant Protoplasts
Center for Genomics and Systems Biology, Department of Biology, New York University
A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.
JoVE General
The Ladder Rung Walking Task: A Scoring System and its Practical Application.
Canadian Centre for Behavioural Neuroscience, Department of Neuroscience, University of Lethbridge
The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.
JoVE Neuroscience
GABA-activated Single-channel and Tonic Currents in Rat Brain Slices
Department of Neuroscience, Uppsala University, Sweden
We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.
JoVE General
Methods for Patch Clamp Capacitance Recordings from the Calyx
National Institute of Neurological Disorders and Stroke, National Institute of Health
We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.
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 General
Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies
We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.
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 General
Transverse Aortic Constriction in Mice
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM)
Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.
JoVE Clinical and Translational Medicine
A Swine Model of Neonatal Asphyxia
1Departments of Pediatrics, Pharmacology and Surgery, University of Alberta, 2Department of Surgery, University of Alberta
Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.
JoVE Immunology and Infection
Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut
1Department of Veterinary Medicine, University of Maryland, 2Department of Entomology, Connecticut Agricultural Experiment Station
Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.
JoVE General
Isolation of Stem Cells from Human Pancreatic Cancer Xenografts
Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine
Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.
JoVE General
IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Department of Immunology, College of Medicine, Mayo Clinic
The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.
JoVE General
Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
1Department of Biological Sciences, University of Alberta, 2Department of Agriculture, Food and Nutrition Sciences, University of Alberta
An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.
JoVE General
Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)
Department of Pathology, Sackler School of Medicine, Tel Aviv University
Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFRα as a surface marker.
JoVE Immunology and Infection
Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice
The Walter and Eliza Hall Institute of Medical Research
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.
JoVE Bioengineering
Adaptation of a Haptic Robot in a 3T fMRI
1Institute for Neural Computation, University of California, 2Department of Radiology, University of California, 3Department of Cognitive Science and Program in Neurosciences, University of California
The adaptation and use of a haptic robot in a 3T fMRI is described.
JoVE Neuroscience
Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices
Department of Biology, University of Texas San Antonio - UTSA
Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.
JoVE General
Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells
Department of Biology, University of Waterloo
Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).
JoVE Immunology and Infection
Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model
1National Health Laboratory Services (NHLS-SA), 2Department of Molecular Medicine and Haematology, University of Witwatersrand, 3Lightcurve Films
Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.
JoVE Clinical and Translational Medicine
Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)
1Department of Neurosurgery, The University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.
JoVE General
Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry
Faculty of Pharmaceutical Sciences, University of British Columbia
Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.
JoVE General
Purification of Progenitors from Skeletal Muscle
The Biomedical Research Centre, University of British Columbia
Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.
JoVE General
Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum
Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.
JoVE Immunology and Infection
Isolation of Brain-infiltrating Leukocytes
Department of Neurology, Mayo Clinic College of Medicine
A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.
JoVE General
Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers
1Tetramer Core Laboratory, Benaroya Research Institute, 2Kwok Laboratory, Benaroya Research Institute, 3Nepom Laboratory, Benaroya Research Institute
This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.
JoVE Immunology and Infection
Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine
1Department of Pediatrics, Emory University, 2Department of Pathology & Laboratory Medicine, Emory University
Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.
JoVE Applied Physics
Echo Particle Image Velocimetry
Mechanical Engineering Department, University of New Hampshire
An echo particle image velocimetry (EPIV) system capable of acquiring two-dimensional fields of velocity in optically opaque fluids or through optically opaque geometries is described, and validation measurements in pipe flow are reported.
JoVE Immunology and Infection
Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center
In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.
JoVE Clinical and Translational Medicine
Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research
1Umeå Centre for Molecular Medicine, Umeå University, 2Cell Transplant Center, Diabetes Research Institute, University of Miami,, 3EMBL-CRG Systems Biology Program, Centre for Genomic Regulation, Catalan Institute of Research and Advanced Studies, 4Dept. of Computing Science, Umeå University
We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic β-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.
JoVE Neuroscience
Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells
1Discipline of Biomedical Science, School of Medical Sciences, Sydney Medical School and Bosch Institute, University of Sydney, 2The MARCS Institute, University of Western Sydney, 3Discipline of Physiology, School of Medical Sciences, Sydney Medical School and Bosch Institute, University of Sydney
This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.
JoVE Neuroscience
The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice
1Institute for Neuroscience, University of Texas at Austin, 2Department of Psychology, University of Texas at Austin, 3Department of Neurology, University of Florida, 4Department of Psychiatry, University of Texas Southwestern Medical Center, 5Department of Neuroscience, McKnight Brain Institute, University of Florida
The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.
JoVE Clinical and Translational Medicine
Stereotactic Radiosurgery for Gynecologic Cancer
1Department of Radiation Oncology, University Hospitals Case Medical Center and Case Western Reserve University School of Medicine, 2Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University Hospitals Case Medical Center and Case Western Reserve University School of Medicine
Stereotactic body radiotherapy (SBRT) involves image-guided, ablative radiation delivered to cancer targets refractory to chemotherapy or to conventional radiation treatment. The robotic-armed Cyberknife SBRT system, using sophisticated target localization, delivers hypofractionated radiation doses capable of sterilizing cancer targets. This article will consider new therapeutic roles of SBRT for gynecological cancers.
JoVE Neuroscience
Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution
Section of Neurobiology, Center for Learning and Memory, The University of Texas at Austin
Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.
More Results...
