Search

Mammalia

Categories

    Protocols in Current Issue
    Instillation of Particulate Suspensions to the Lungs
    Authors:  Etsushi Kuroda, Yasuo Morimoto and Ken J Ishii, date: 11/20/2017, view: 98, Q&A: 0
    Inhaled fine particulates are thought to cause chronic pulmonary inflammation through the deposition of particulates into the lungs. To investigate the effect of fine particulates on the lungs, instillation of suspension of particulates into the ...
    Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay
    Authors:  Nathanael P. Cottam and Daniel Ungar, date: 11/20/2017, view: 102, Q&A: 0
    Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable ...
    In vitro Homeostatic Proliferation of Human CD8 T Cells
    Authors:  Hossam A. Abdelsamed, Caitlin C. Zebley and Ben Youngblood, date: 11/20/2017, view: 68, Q&A: 0
    Long-lived T-cell–mediated immunity requires persistence of memory T cells in an antigen-free environment while also maintaining a heightened capacity to recall effector functions. Such antigen-independent homeostatic proliferation is mediated in ...
    Quantification of Trypanosoma cruzi in Tissue and Trypanosoma cruzi Killing Assay
    Authors:  Hisako Kayama, Shoko Kitada and Kiyoshi Takeda, date: 11/20/2017, view: 58, Q&A: 0
    Infection with Trypanosoma cruzi causes Chagas disease. The methods provided here allow for the quantification of T. cruzi in the liver, heart, and blood of intraperitoneally-infected mice and analysis of the killing activity of ...
    Cell-free Generation of COPII-coated Procollagen I Carriers
    Authors:  Lin Yuan, Satoshi Baba, Kanika Bajaj and Randy Schekman, date: 11/20/2017, view: 132, Q&A: 0
    The aim of this protocol is to generate COPII-coated procollagen I (PC1) carriers in a cell-free reaction. The COPII-coated PC1 carriers were reconstituted from donor membrane, cytosol, purified recombinant COPII proteins, and nucleotides. This ...
    Obtaining Multi-electrode Array Recordings from Human Induced Pluripotent Stem Cell–Derived Neurons
    Authors:  Xiaohong Xu, Carola I. Radulescu, Kagistia Hana Utami and Mahmoud A. Pouladi , date: 11/20/2017, view: 112, Q&A: 0
    Neuronal electrical properties are often aberrant in neurological disorders. Human induced pluripotent stem cells (hiPSCs)-derived neurons represent a useful platform for neurological disease modeling, drug discovery and toxicity screening in vitro ...
    A Streamlined Method for the Preparation of Gelatin Embedded Brains and Simplified Organization of Sections for Serial Reconstructions
    Authors:  Andrew W. Liu, Sho Aoki and Jeffery R. Wickens, date: 11/20/2017, view: 163, Q&A: 0
    Gelatin embedding of whole brains for sectioning is a critical procedure used in neuroscience to ensure all morphological and spatial details are preserved intact. Here, we describe an inexpensive, reproducible and efficient means to embed ...
    Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells
    Authors:  Maria Fe Lanfranco, David J. Loane, Italo Mocchetti, Mark P. Burns and Sonia Villapol, date: 11/20/2017, view: 120, Q&A: 0
    Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that ...
    Organotypic Brain Cultures: A Framework for Studying CNS Infection by Neurotropic Viruses and Screening Antiviral Drugs
    According to the World Health Organization (WHO), at least 50% of emerging viruses endowed with pathogenicity in humans can infect the Central Nervous System (CNS) with induction of encephalitis and other neurologic diseases (Taylor et al., ...
    Bioluminescence Resonance Energy Transfer (BRET) Assay for Determination of Molecular Interactions in Living Cells
    The bioluminescence resonance energy transfer (BRET) assay can be used as an indicator of molecular approximation and/or interaction. A significant resonance energy transfer signal is generated when the acceptor, having the appropriate spectral ...
    Detection of Membrane Protein Interactions by Cell-based Tango Assays
    The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV ...
    Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization
    Pulldown assay is a conventional method to determine protein-protein interactions in vitro. Expressing a protein of interest with two different tags allows testing whether both versions can be captured via one of the two tags as ...
    γ-Secretase Epsilon-cleavage Assay
    γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang et al., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are ...
    An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins
    Authors:  Thomas J Macartney, Gopal P Sapkota and Luke J Fulcher, date: 11/20/2017, view: 84, Q&A: 0
    We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) (Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a ...
    Instillation of Particulate Suspensions to the Lungs
    Authors:  Etsushi Kuroda, Yasuo Morimoto and Ken J Ishii, date: 11/20/2017, view: 98, Q&A: 0
    [Abstract] Inhaled fine particulates are thought to cause chronic pulmonary inflammation through the deposition of particulates into the lungs. To investigate the effect of fine particulates on the lungs, instillation of suspension of particulates into the lungs is required. This protocol describes direct injection of suspensions of fine particulates into ...
    Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay
    Authors:  Nathanael P. Cottam and Daniel Ungar, date: 11/20/2017, view: 102, Q&A: 0
    [Abstract] Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was ...
    In vitro Homeostatic Proliferation of Human CD8 T Cells
    Authors:  Hossam A. Abdelsamed, Caitlin C. Zebley and Ben Youngblood, date: 11/20/2017, view: 68, Q&A: 0
    [Abstract] Long-lived T-cell–mediated immunity requires persistence of memory T cells in an antigen-free environment while also maintaining a heightened capacity to recall effector functions. Such antigen-independent homeostatic proliferation is mediated in part by the common gamma-chain cytokines IL-7 and IL-15. To further explore the mechanisms governing ...
    Quantification of Trypanosoma cruzi in Tissue and Trypanosoma cruzi Killing Assay
    Authors:  Hisako Kayama, Shoko Kitada and Kiyoshi Takeda, date: 11/20/2017, view: 58, Q&A: 0
    [Abstract] Infection with Trypanosoma cruzi causes Chagas disease. The methods provided here allow for the quantification of T. cruzi in the liver, heart, and blood of intraperitoneally-infected mice and analysis of the killing activity of the cells infected with T. cruzi in vitro.
    Cell-free Generation of COPII-coated Procollagen I Carriers
    Authors:  Lin Yuan, Satoshi Baba, Kanika Bajaj and Randy Schekman, date: 11/20/2017, view: 132, Q&A: 0
    [Abstract] The aim of this protocol is to generate COPII-coated procollagen I (PC1) carriers in a cell-free reaction. The COPII-coated PC1 carriers were reconstituted from donor membrane, cytosol, purified recombinant COPII proteins, and nucleotides. This protocol describes the preparation of donor membrane and cytosol, the assembly of the reaction, and the ...
    Obtaining Multi-electrode Array Recordings from Human Induced Pluripotent Stem Cell–Derived Neurons
    Authors:  Xiaohong Xu, Carola I. Radulescu, Kagistia Hana Utami and Mahmoud A. Pouladi , date: 11/20/2017, view: 112, Q&A: 0
    [Abstract] Neuronal electrical properties are often aberrant in neurological disorders. Human induced pluripotent stem cells (hiPSCs)-derived neurons represent a useful platform for neurological disease modeling, drug discovery and toxicity screening in vitro. Multi-electrode array (MEA) systems offer a non-invasive and label-free platform to record ...
    A Streamlined Method for the Preparation of Gelatin Embedded Brains and Simplified Organization of Sections for Serial Reconstructions
    Authors:  Andrew W. Liu, Sho Aoki and Jeffery R. Wickens, date: 11/20/2017, view: 163, Q&A: 0
    [Abstract] Gelatin embedding of whole brains for sectioning is a critical procedure used in neuroscience to ensure all morphological and spatial details are preserved intact. Here, we describe an inexpensive, reproducible and efficient means to embed post-fixed brains ready for sectioning in gelatin within a week’s time. The sections obtained are ...
    Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells
    Authors:  Maria Fe Lanfranco, David J. Loane, Italo Mocchetti, Mark P. Burns and Sonia Villapol, date: 11/20/2017, view: 120, Q&A: 0
    [Abstract] Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope® ...
    Organotypic Brain Cultures: A Framework for Studying CNS Infection by Neurotropic Viruses and Screening Antiviral Drugs
    [Abstract] According to the World Health Organization (WHO), at least 50% of emerging viruses endowed with pathogenicity in humans can infect the Central Nervous System (CNS) with induction of encephalitis and other neurologic diseases (Taylor et al., 2001; Olival and Daszak, 2005). While neurological diseases are progressively documented, the ...
    Bioluminescence Resonance Energy Transfer (BRET) Assay for Determination of Molecular Interactions in Living Cells
    [Abstract] The bioluminescence resonance energy transfer (BRET) assay can be used as an indicator of molecular approximation and/or interaction. A significant resonance energy transfer signal is generated when the acceptor, having the appropriate spectral overlap with the donor emission, is approximated with the donor. In the example provided, proteins ...