Protocols in Current Issue
    CD8 T Cell Virus Inhibition Assay Protocol

    The human immunodeficiency virus (HIV)-1 viral inhibition assay (VIA) measures CD8+ T cell-mediated inhibition of HIV replication in CD4+ T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. Different VIAs that differ in length of CD8:CD4 T cell culture periods (6–13 days), purity of CD4 cultures

    A Combinatorial in-silico, in-vitro and in-vivo Approach to Quantitatively Study Peptide Induced MHC Stability
    Authors:  Roman Sarkar, Yashu Sharma, Ayush Jain, Azeez Tehseen, Sudhakar Singh and Sharvan Sehrawat, date: 12/20/2021, view: 996, Q&A: 0

    Here, we describe a combinatorial approach in reverse vaccinology to identify immunogenic class I major histocompatibility complex (MHC) displayed epitopes derived from a morbillivirus named pestes des petits ruminants (PPRV). The protocol describes an in silico prediction of immunogenic epitopes using an IEDB tool. The predicted peptides were

    Isolation and Quantification of Mouse γδT-cells in vitro and in vivo
    Authors:  Isha Rana, Krithika Badarinath, Ravindra K. Zirmire and Colin Jamora, date: 09/05/2021, view: 1742, Q&A: 0

    The skin plays an important role in protecting the body from pathogens and chemicals in the external environment. Upon injury, a healing program is rapidly initiated and involves extensive intercellular communication to restore tissue homeostasis. The deregulation of this crosstalk can lead to abnormal healing processes and is the foundation of

    One-step White Blood Cell Extracellular Staining Method for Flow Cytometry

    Flow cytometry is a powerful analytical technique that is increasingly used in scientific investigations and healthcare; however, it requires time-consuming, multi-step sample procedures, which limits its use to specialized laboratories. In this study, we propose a new universal one-step method in which white blood cell staining and red blood cell

    Multi-color Flow Cytometry for Comprehensive Analysis of the Tumor Immune Infiltrate in a Murine Model of Breast Cancer
    Authors:  Ana S. Almeida, Miriam R. Fein and Mikala Egeblad, date: 06/05/2021, view: 2942, Q&A: 0

    Flow cytometry is a popular laser-based technology that allows the phenotypic and functional characterization of individual cells in a high-throughput manner. Here, we describe a detailed procedure for preparing a single-cell suspension from mammary tumors of the mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) and analyzing these cells by

    A Simple and Robust Protocol for in vitro Differentiation of Mouse Non-pathogenic T Helper 17 Cells from CD4+ T Cells
    Authors:  Siwen Kang, Ruohan Wu and Ruoning Wang, date: 05/20/2021, view: 2926, Q&A: 0

    Functional and mechanistic studies of CD4+ T cell lineages rely on robust methods of in vitro T cell polarization. Here, we report an optimized protocol for in vitro differentiation of a mouse non-pathogenic T helper 17 (TH17) cell lineage. Most of the previously established protocols require irradiated splenocytes as artificial antigen presenting

    Flow-cytometric Detection of Low-level Reactive Oxygen Species in Cell Lines and Primary Immune Cells
    Authors:  Kevin Bode, Corinna Link, Peter H. Krammer and Heiko Weyd, date: 09/05/2020, view: 3886, Q&A: 0
    [Abstract] Depending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation ...
    Flow Cytometry Analysis and Fluorescence-activated Cell Sorting of Myeloid Cells from Lung and Bronchoalveolar Lavage Samples from Mycobacterium tuberculosis-infected Mice
    Authors:  Alissa C. Rothchild, Dat Mai, Alan Aderem and Alan H. Diercks, date: 05/20/2020, view: 3958, Q&A: 0
    [Abstract] Mycobacterium tuberculosis (Mtb) is transmitted by aerosol and can cause serious bacterial infection in the lung that can be fatal if left untreated. Mtb is now the leading cause of death worldwide by an infectious agent. Characterizing the early events of in vivo infection following aerosol challenge is critical for ...
    Assessing in vitro and in vivo Trogocytosis By Murine CD4+ T cells
    Authors:  Jim Reed and Scott A. Wetzel, date: 05/05/2020, view: 3033, Q&A: 0
    [Abstract] Recognition of antigens by lymphocytes (B, T, and NK) on the surface of an antigen-presenting cell (APC) leads to lymphocyte activation and the formation of an immunological synapse between the lymphocyte and the APC. At the immunological synapse APC membrane and associated membrane proteins can be transferred to the lymphocyte in a process called ...
    Characterization of Immunological Niches within Peyer’s Patches by ex vivo Photoactivation and Flow Cytometry Analysis
    Authors:  Adi Biram and Ziv Shulman, date: 03/20/2020, view: 3103, Q&A: 0
    [Abstract] T follicular helper (Tfh) cells regulate B cell selection for entry into the germinal center (GC) reaction or for differentiation into antibody forming cells. This process takes place at the border between the T and B zones in lymphoid organs and involves physical contacts between T and B cells. During these interactions, T cells endow the B cells ...

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