Protocols in Current Issue
    Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2
    Authors:  Seeyoung Choi and Paul E. Love, date: 01/05/2018, view: 204, Q&A: 0
    [Abstract] Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the ...
    Oral Microbiome Characterization in Murine Models
    [Abstract] The oral microbiome has been implicated as a trigger for immune responsiveness in the oral cavity, particularly in the setting of the inflammatory disease periodontitis. The protocol presented here is aimed at characterizing the oral microbiome in murine models at steady state and during perturbations of immunity or physiology. Herein, we describe ...
    Generation of Busulfan Chimeric Mice for the Analysis of T Cell Population Dynamics
    Authors:  Thea Hogan, Andrew Yates and Benedict Seddon, date: 12/20/2017, view: 249, Q&A: 0
    [Abstract] This protocol was developed to generate chimeric mice in which T lymphocytes could be stratified by age on the basis of congenic marker expression. The conditioning drug busulfan is used to ablate host haematopoietic stem cells while leaving the peripheral immune system intact. Busulfan treatment is followed by bone marrow transplantation (BMT), ...
    Analysing Temporal Dynamics of T Cell Division in vivo Using Ki67 and BrdU Co-labelling by Flow Cytometry
    Authors:  Thea Hogan, Andrew Yates and Benedict Seddon, date: 12/20/2017, view: 349, Q&A: 0
    [Abstract] This protocol was developed to increase the richness of information available from in vivo T cell proliferation studies. DNA labelling techniques such as BrdU incorporation allow precise control of label administration and withdrawal, so that the division history of a population can be tracked in detail over long timeframes (days-weeks). ...
    Immunogold Electron Microscopy of the Autophagosome Marker LC3
    Authors:  Domenico Mattoscio, Andrea Raimondi, Carlo Tacchetti and Susanna Chiocca, date: 12/20/2017, view: 347, Q&A: 0
    [Abstract] Even though autophagy was firstly observed by transmission electron microscopy already in the 1950s (reviewed in Eskelinen et al., 2011), nowadays this technique remains one of the most powerful systems to monitor autophagic processes. The autophagosome, an LC3-positive double membrane structures enclosing cellular materials, represents ...
    Mouse Model of Immune Complex-mediated Vasculitis in Dorsal Skin and Assessment of the Neutrophil-mediated Tissue Damage
    [Abstract] Neutrophils are the most abundant leukocytes in the blood. In the recent decades, their crucial roles in host defense, immune regulation and tissue damage have been studied in a deeper dimension. In this protocol, we described a mouse model of immune complex-mediated vasculitis in the dorsal skin induced by Arthus reaction, and the subsequent ...
    Lentiviral Knockdown of Transcription Factor STAT1 in Peromyscus leucopus to Assess Its Role in the Restriction of Tick-borne Flaviviruses
    Authors:  Adaeze O. Izuogu and R. Travis Taylor, date: 12/05/2017, view: 407, Q&A: 0
    [Abstract] Cellular infection with tick-borne flaviviruses (TBFVs) results in activation of the interferon (IFN) signaling pathway and subsequent upregulation of numerous genes termed IFN stimulated genes (ISGs) (Schoggins et al., 2011). Many ISGs function to prevent virus pathogenesis by acting in a broad or specific manner through protein-protein ...
    Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells
    Authors:  Florent Ubelmann, Tatiana Burrinha and Claudia Guimas Almeida, date: 12/05/2017, view: 425, Q&A: 0
    [Abstract] The established primary trigger of Alzheimer’s disease’s is β-amyloid (Aβ) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane (Koo et al., 1996; Ubelmann et ...
    Drosophila Model of Leishmania amazonensis Infection
    Authors:  Kendi Okuda and Neal Silverman, date: 12/05/2017, view: 400, Q&A: 0
    [Abstract] This protocol describes how to generate and harvest antibody-free L. amazonensis amastigotes, and how to infect adult Drosophila melanogaster with these parasites. This model recapitulates key aspects of the interactions between Leishmania amastigotes and animal phagocytes.
    Instillation of Particulate Suspensions to the Lungs
    Authors:  Etsushi Kuroda, Yasuo Morimoto and Ken J Ishii, date: 11/20/2017, view: 651, 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 ...