Protocols in Current Issue
    Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor
    Authors:  Rubén Martínez-Barricarte, Xiao-Fei Kong and Jean-Laurent Casanova, date: 06/05/2019, view: 523, Q&A: 0
    [Abstract] The recent discovery of human signal peptide peptidase-like 2a (SPPL2a) deficiency in humans revealed the toxicity associated with the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF), for certain type of dendritic cells (cDC2). We developed a two-step protocol for monitoring the accumulation of this molecule in different ...
    Induced Germinal Center B Cell Culture System
    Authors:  Kei Haniuda and Daisuke Kitamura, date: 02/20/2019, view: 2061, Q&A: 0
    [Abstract] The germinal center (GC) is the site where B cells undergo clonal expansion, affinity-based selection, and differentiation into memory B cells or plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells in vivo ...
    Assessment of Humoral Alloimmunity in Mixed Lymphocyte Reaction
    Authors:  Georgios Pissas and Theodoros Eleftheriadis, date: 01/20/2019, view: 1742, Q&A: 1
    [Abstract] Humoral alloimmunity remains a significant and unresolved problem that constrains allograft survival. Thus, there is a need for the development of an easy, preferably non-radioactive, and inexpensive protocol for assessing the effect of various drug treatments on humoral alloimmunity. In order to satisfy this demand, we developed such a protocol ...
    Integrin Dependent RhoB Activation Assay Using Leukocytes
    Authors:  Janne Lehtonen, Katarzyna Potrzebowska, Malin Samuelsson and Lena Svensson, date: 12/05/2018, view: 775, Q&A: 0
    [Abstract] To be able to migrate, leukocyte needs to re-use its adhesion molecules to move forward. These adhesion molecules are called integrins and are intracellularly transported via endocytosis and exocytosis in order to translocate to a new site on the cell membrane. The intracellular transportation is regulated by different small GTPases including ...
    Mouse CD8+ T Cell Migration in vitro and CXCR3 Internalization Assays
    Authors:  Rosa Barreira da Silva and Matthew L. Albert, date: 03/20/2017, view: 8124, Q&A: 0
    [Abstract] Chemokines are molecules that regulate the positioning of cells during homeostasis and inflammation. CXCL10 is an interferon-induced chemokine that attracts cells that express the chemokine receptor CXCR3 on their surface. CXCL10 expression is often induced upon inflammation and guides lymphocytes, such as T and NK cells, into the injured tissues. ...
    In vitro Assessment of Immunological Synapse Formation by Flow Cytometry
    Authors:  Bo-Ra Na and Chang-Duk Jun, date: 03/20/2016, view: 9526, Q&A: 2
    [Abstract] In adaptive immune system, formation of immunological synapse between T cells and antigen presenting cells (dendritic cells, B cells, and macrophages) or target cells (tumor cells and viral-infected cells) is critical for the execution of T cell immune responses via cytokine secretion or direct killing activity. Here, we describe the practical ...
    Mouse BMDC-dependent T Cell Polarization Assays
    Author:  Vijaya Satchidanandam, date: 02/05/2016, view: 7811, Q&A: 0
    [Abstract] In response to exposure to antigen, T cells whose T cell receptor (TCR) are capable of recognizing the self MHC-antigen derived peptide complex, respond to the antigen and differentiate into one of several subsets, namely TH1, TH2, TH17, Treg, etc. characterized by the signature cytokine they secrete, namely IFN-γ, IL-4, IL-17 or IL-10, ...
    Protocol-In vitro T Cell Proliferation and Treg Suppression Assay with Celltrace Violet
    Authors:  Kristofor K. Ellestad and Colin C. Anderson, date: 01/05/2016, view: 17684, Q&A: 0
    [Abstract] Measurement of the incorporation of radionuclides such as 3H-thymidine is a classical immunological technique for assaying T cell proliferation. However, such an approach has drawbacks beyond the inconvenience of working with radioactive materials, such as the inability of bulk radionuclide incorporation measurements to accurately ...
    TCRβ Clonotype Analysis of EBV and CMV-specific Human CD8+ T Cells
    Authors:  Nening M. Nanlohy, Dan Koning, Esther D. Quakkelaar and Debbie van Baarle, date: 10/05/2015, view: 4540, Q&A: 0
    [Abstract] This protocol describes the quantification of all expressed T-cell antigen receptor (TCR) gene products within sorted (by flow cytometry) EBV and CMV-specific memory CD8+ T-cell populations using a template-switch anchored reverse transcription polymerase chain reaction (RT-PCR).
    Isolation and in vivo Transfer of Antigen Presenting Cells
    Authors:  Pooja Arora, Shalu Sharma Kharkwal and Steven A. Porcelli, date: 10/05/2014, view: 8092, Q&A: 0
    [Abstract] Transfer of antigen presenting cells in vivo is a method used by immunologists to examine the potency of antigen presentation by a selected population of cells. This method is most commonly used to analyze presentation of protein antigens to MHC class I or II restricted T cells, but it can also be used for studies of nonconventional ...

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