SUBJECT
Methods in Immunology II
practical
master
4
Semesters 1-4
Autumn/Spring semester
Blocked practicals:
● Investigation of antigen presentation in a murine model system (mouse B lymphoma cell as APC plus influenza virus-specific TH cells). I. Detection of the early (0-5 min) calcium signal of TH-cells upon antigen stimulation.
● Investigation of antigen presentation in a murine model system (mouse B lymphoma cell as APC plus influenza virus-specific TH cells). II. Detection of the late (24-48 hrs) cytokine production of TH cells.
● Activation of B cells by crosslinking of the surface immunoglobulin (BCR). I. Analysis of the early Ca2+-response. Comparison of the features of calcium response curves shown by B and T cells.
● Activation of B cells by crosslinking of the surface immunoglobulin (BCR). II. Detection and analysis of the rapid, receptor-mediated tyrosine-phosphorylation.
● Separation of the T and B lymphocytes: the „panning” and the „complement mediated lysis” methods. Testing the efficiency of separation by flow cytometry (FACS) technique.
● Detection of changes in MHC-II and costimulator (B7) expression of murine spleen cells upon activation with LPS (Flow Cytometry, FACS).
● Detection of the degranulation following mast cell activation.
● Fluorescence confocal microscopy (practical & demonstrations) I. Detection of protein localization using immunoflorescence labeling, investigation of membrane rafts in lymphocytes by the colocalization method.
● Detection of programmed cell death (apoptosis) in lymphocytes: measurement of mitochondrial membrane potential by flow cytometry
● Detection of late apoptotic events: DNA-fragmentation and membrane blebbing by flow and imaging cytometric techniques
●Analysis of macrophage phagocytosis by digital imaging microscopy.
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Gerard Meurant: Immunological Methods, Elsevier, 2012