Principles and applications of flow cytometry. Strengths and limits in comparison with other technologies.
Basic principles: light-particle interaction, diffusion, diffraction, fluorescence.
Instrumentation: optical, fluidic, electronic and software components.
Fluorescent probes, general aspects, excitation and emission spectra, FRET, tandem fluorochromes. Spillover and compensation.
Sample preparation, single-cell suspension, viability, fixation, permeabilization, sample preservation.
Staining intracellular and intranuclear cell surface molecules.
Multi-parametric analyses. Principles of sample analysis, evaluation of cell viability, off-line compensation, staining controls (blank, isotype and isoclonic controls, fluorescence minus one. Gating strategies. Criteria for evaluating positivity (positive / negative, low / high, continuous markers); percentages of positive cells, mean and median fluorescence intensity.
Representation of cytometric data, histograms, dot plots, density and contour plots.
Cell cycle analysis, cell proliferation, apoptosis, cell activation (calcium flow, protein phosphorylation).
Analysis of intracellular and nuclear proteins and of RNA. Measurement of analytes in culture supernatants and biological fluids. Comparisons with western blot, RT-PCR and ELISA.
Phenotyping of leukocytes; identification of lymphocyte subpopulations.
Studying lymphocyte development and maturation by flow cytometry.
Evaluation of phagocytosis by flow cytometry.
Examples of non-biomedical applications.
Principles of cell sorting by flow cytometry.
Exercises with sample analysis.
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