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Department of Clinical Immunology Wroclaw Medical University ul. Chałubińskiego 5, 50-368 Wrocław tel.: 71 784 17 40, faks: 71 784 04 17 e-mail: [email protected] Head: prof. dr hab. Marek Jutel IMMUNOLOGY COURSE DETAILED ISSUES Faculty of Medicine AY 2014/2015 Class 2. Cellular immunity – cell isolation assays, cell phenotype assessment. Flow cytometry. Issues: 1. Type of biologic material for immunophenotyping (blood, organic fluids, tissue samples). 2. Immune cells isolation methods: - density gradient based isolation (method principles, density gradient characteristics) - immunoadsorbent assays (panning) - magnetic-activated cell sorting (MACS); positive selection, negative selection - fluorescence-activated cell sorting (FACS) 3. Terms: cell genotype, cell phenotype; immunophenotype. 4. Immune cell surface markers (CD molecules). 5. Flow cytometry (FC). - type of samples - basics of the technique - basic parameters assessed: forward scattering (FSC), side scattering (SSC), fluorescence emission - FC data presentation - types of plots, data interpretation principles, gating; examples - FC controls examples (autofluorescence control, isotype control) – short description. 6. Application of flow cytometry. - cell immunophenotyping: lymphocyte subpopulations - diagnostics and monitoring in hematology diseases and immunodeficiences - other: cell proliferation, intracellular cytokines production, DNA analyzes, cell apoptosis, other. Practical part: 1. Density gradient peripheral blood mononuclear cell (PBMC) isolation (Gradisol-L) 2. Lymphocyte viability assessment test – trypan blue dying; the light miscroscope assessment Recommended literature (FC): http://probes.invitrogen.com/resources/education/nt 1