Download immunology course detailed issues -2

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
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