SCIENCE EDUCATION > Advanced Biology

Immunology

This collection covers many staple techniques of immunology labs, including the labeling and sorting of immune cells. It also demonstrates proliferation methods for immune cells and antibodies, as well as common assays for immune activity including ELISA. Finally, it demonstrates staining and imaging of immune tissue and cell samples.

  • Immunology

    17:07
    Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS): Isolation of Splenic B Lymphocytes

    Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 2 INSERM U1223, Paris, France 3 Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France 4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France The overall function of the immune system is to defend the body against infectious organisms and other invaders. White blood cells, or leukocytes, are the key players of the immune system. Upon infection, they are activated and initiate an immune response. Leukocytes can be divided into various sub-populations (e.g., myeloid cells, lymphocytes, dendritic cells) based on different parameters that can be biological, physical, and/or functional (e.g., size, granularity, and secretion). One way to characterize leukocytes is through their surface proteins, which are mainly receptors. Each leukocyte population expresses a specific combination of receptors (e.g., cytotoxic, activating, migration receptors) that can define subsets among populations. As the immune system encompasses a wide range of cell populations, it is essential to characterize them to decipher their participation in the immune resp

  • Immunology

    11:35
    Magnetic Activated Cell Sorting (MACS): Isolation of Thymic T Lymphocytes

    Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 2 INSERM U1223, Paris, France 3 Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France 4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France Defence against pathogens depends on surveillance by the immune system. This system is complex and comprises many cell types, each one with specific functions. This complex composition enables immune responses to a large diversity of pathogens and injuries. Adaptive immunity allows specific responses against specific pathogens. The majority of cells responsible for this type of immunity are the lymphocytes (B cells and T cells). Usually, B cells respond to extracellular infections (such as bacterial infections), and T cells respond to intracellular infections (such as viral infections). The different types of cells in lymphocyte populations can be characterized by the combination of cell surface proteins they express and/or by a panel of secreted cytokines. Magnetic sorting allows enrichment of targeted cell populations using magnetic properties and expression of one or several cell surface proteins (1, 2). This

  • Immunology

    14:21
    ELISA Assays: Indirect, Sandwich, and Competitive

    Source: Whitney Swanson1,2, Frances V. Sjaastad2,3, and Thomas S. Griffith1,2,3,4 1 Department of Urology, University of Minnesota, Minneapolis, MN 55455 2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 3 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455 Enzyme-linked immunosorbent assay (ELISA) is frequently used to measure the presence and/or concentration of an antigen, antibody, peptide, protein, hormone, or other biomolecule in a biological sample. It is extremely sensitive, capable of detecting low antigen concentrations. The sensitivity of ELISA is attributed to its ability to detect the interactions between a single antigen-antibody complex (1). Moreover, the inclusion of an enzyme-conjugated antigen-specific antibody permits the conversion of a colorless substrate into a chromogenic or fluorescent product that can be detected and easily quantitated by a plate reader. When compared to the values generated by titrated amounts of a known antigen of interest, the concentration of the same antigen in the experimental samples can be determined. Different ELISA protocols have been adapted to measure antigen concentrations in a variety of experimental samples, but they all have th

  • Immunology

    11:53
    ELISPOT Assay: Detection of IFN-γ Secreting Splenocytes

    Source: Tonya J. Webb1 1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201

    ELISPOT is a standardized, reproducible assay used to detect cellular immune responses. The assay utilizes an enzyme-linked immunosorbent assay (ELISA)- based method to detect single-cell immune responses which can be visualized by spots, hence the name ELISPOT. ELISPOT was first described in 1983, by Czerkinsky, as a method of enumerating the number of B cell hybridomas producing antigen-specific immunoglobulins (1). The same group further developed the assay to measure the frequency of cytokine producing T lymphocytes. Now ELISPOT has become a gold standard for measuring antigen-specific T cell immunity in clinical trials and vaccine candidates. For example, after vaccination or during an infection, plasma cells and memory B cells secrete antibodies that provide protection. Typically, these B cell responses are assessed by measuring serum titers of antigen-specific antibodies. However, this type of analysis, typically measured by ELISA, may not include memory B cells, which can be present even in the absence of detectable serum antibody levels. Furthermore, it has been well-established that circulating memory B cells are important for the rapid and protective antibody response observed fol

  • Immunology

    13:23
    Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy

    Source: Michael S. Lee1 and Tonya J. Webb1 1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201

    Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques used to visualize the expression and localization of specific antigens using antibodies. The first published use of IHC was in 1941 when Albert Coons used the technique to visualize the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus (1). The name, immunohistochemistry, is derived from the roots "immuno-," in reference to antibodies, and "histo-," in reference to the tissue sections used in IHC. The root "cyto-" in immunocytochemistry highlights the key difference between ICC and IHC. Whereas IHC uses sections of whole tissue, ICC uses cells that have been isolated from tissue or grown in culture. The difference in samples used means sample preparation technically differs between IHC and ICC, but otherwise the protocols for ICC and IHC are identical and one will find the terms are frequently used interchangeably. In both IHC and ICC, antibodies with either chemical or fluorescent tags, such as peroxidase or rhodamine, respectively, are used to visualize the distribution of any antigen of interest through specifi

  • Immunology

    13:20
    Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

    Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455 Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins. Several approaches have been tak

  • Immunology

    09:55
    Immunofluorescence Microscopy: Immunofluorescence Staining of Paraffin-Embedded Tissue Sections

    Source: Thomas Chaffee1, Thomas S. Griffith2,3,4, and Kathryn L. Schwertfeger1,3,4 1 Department of Lab Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455 2 Department of Urology, University of Minnesota, Minneapolis, MN 55455 3 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455 4 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 Pathologic analyses of tissue sections can be used to obtain a better understanding of normal tissue structure and contribute to our understanding of mechanisms of disease. Tissue biopsies, either from patients or from experimental in vivo models, are often preserved by fixing in formalin or paraformaldehyde and embedding in paraffin wax. This allows for long-term storage and for the tissues to be sectioned. Tissues are cut into thin (5 µm) sections using a microtome and the sections are adhered to glass slides. The tissues sections can be stained with antibodies, which allow for the detection of specific proteins within the tissue sections. Staining with antibodies conjugated to fluorophores (also known as fluorochromes) - compounds that emit light at specific wavelengths when excited by a laser - is known as immunofluorescence. The ability to detect proteins within a section can provide information such as cell type heterogeneity within the

  • Immunology

    13:13
    Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts

    Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1 1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201 2 Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland 21201

    Confocal fluorescence microscopy is an imaging technique that enables increased optical resolution as compared to conventional "wide-field" epifluorescence microscopy. Confocal microscopes are able to achieve improved x-y optical resolution through "laser scanning"- typically a set of voltage-controlled mirrors (galvanometer or "galvo" mirrors) that direct laser-illumination to each pixel of the specimen at a time. More importantly, confocal microscopes achieve superior z-axial resolution by using a pinhole to remove out of focus light originating from locations that are not in the z-plane being scanned, thus enabling the detector to collect data from a specified z-plane. Because of the high z-resolution achievable in confocal microscopy, it is possible to collect images from a series of z-planes (also-called z-stack) and construct a 3D image through software. Before discussing the mechanism of a confocal microscope, it is important to consider how a sample inter

  • Immunology

    13:46
    Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

    Source: Susannah C. Shissler1, Tonya J. Webb1 1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201

    Immunoprecipitation (IP, also known as a 'pull-down' assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word "immuno" refers to the use of an antibody while the word "precipitation" refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subseq

  • Immunology

    10:56
    Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

    Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 2 INSERM U1223, Paris, France 3 Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France 4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism's life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs. Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA

  • Immunology

    11:03
    Adoptive Cell Transfer: Introducing Donor Mouse Splenocytes to a Host Mouse and Assessing Success via FACS

    Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 2 INSERM U1223, Paris, France 3 Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France 4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France Adoptive cell transfer is a method for introducing cells into a patient or study organism in order to treat a disease or study a biological process, such as haematopoiesis. Aims of adoptive transfer are various; it can be used in fundamental biology as well as in medical sciences (1, 2). In mouse models, migration and distribution of transferred cells can be studied and followed by a tracking system (cell surface marker, staining by CFSE, etc.). In cancer studies on mouse models, transfer of specific cell populations can be used as experimental treatment against tumors. Another example for this technique is creation of chimeric mice by transfer of bone marrow cells to irradiated mice or mice with a severe immunodeficiency phenotype. This mouse model can be used to assess the impact of gene deletion on a specific cell population for instance. Transfer of bone borrow cells is also used in human medical treatment. When pat

  • Immunology

    11:47
    Assay for Cell Death: Chromium Release Assay of Cytotoxic Ability

    Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455 One of the main functions of the cells of the immune system is to remove target cells that have been infected with viruses or have undergone transformation into a tumor cell. In vitro assays for measuring the cytotoxic capacity of immune cells have been a staple in laboratories for many years. These assays have been used to determine the ability of T cells, NK cells, or any other immune cell to kill target cells in an antigen-specific or -nonspecific manner. Death ligands (e.g., Fas ligand or TRAIL), cytokines (e.g., IFNg or TNF), or cytotoxic granules (i.e., perforin/granzyme B) expressed by effector cells are some ways in which target cell death can be induced. With the explosion in tumor immunotherapy research in recent years, there is growing interest in finding agents to increase the cytotoxic activity of immune cells to improve patient outcomes. Conversely, some diseases are hallmarked by the overexuberant activity o

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