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Lecturer(s)
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Doležel Jaroslav, prof. Ing. DrSc.
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Course content
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Classification of cytometric techniques, quantitative cell characterization, and imaging cytometry. Principles of laminar flow, sheath fluid, hydrodynamic and acoustic focusing, microfluidics on a chip. Excitation light sources, optical filters, scatter and fluorescence detectors, spatial laser separation. Photodiodes, photomultipliers, light pulse parameters, doublet discrimination, data digitization, and data formats. Fluorescence, Stokes shift, fluorescence compensation, and spectral cytometry (spectral unmixing). Principles and modes of cell sorting, purity, yield, sperm sorting, and chromosome purification. Protoplast analysis, cell viability, reporter genes, somatic hybridization, and chloroplast study. Nuclear isolation, DNA fluorochromes, standardization of DNA content measurement, and chromosome sorting for genomics. Phytoplankton analysis, phytopathology, Luminex technology and multiplex assays, apoptosis diagnostics. Immunophenotyping, CD nomenclature, measurement of physiological parameters, mass cytometry, in vivo flow cytometry, and photoacoustic effect.
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Learning activities and teaching methods
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Lecture, Monologic Lecture(Interpretation, Training), Dialogic Lecture (Discussion, Dialog, Brainstorming)
- Attendace
- 12 hours per semester
- Homework for Teaching
- 12 hours per semester
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Learning outcomes
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The aim of the course is to provide students with a comprehensive overview of the physical, optical, and technological principles of flow, spectral, and mass cytometry, including advanced cell and chromosome sorting techniques. Graduates will understand signal detection and data processing, and develop the ability to critically select and apply cytometric methods in plant biology, ecology, biotechnology, and biomedical research.
Upon successful completion of the course, students will be able to: - Explain the physical and technical principles of fluidics, optical paths, signal detection, and data digitization in cytometry. - Compare the principles of conventional fluorescence compensation with the spectral unmixing process in spectral cytometry. - Describe the mechanisms, modes, and critical parameters of cell and chromosome sorting. - Characterize methodological procedures for nuclear DNA analysis, genome size estimation, and cell cycle analysis in plant and animal models. - Select appropriate cytometric fluorochrome labeling and methods to address specific questions in ecology, phytopathology, and biomedicine. - Interpret multidimensional cytometric data and standardize the measurement process. - Critically evaluate scientific articles utilizing cytometric and sorting techniques.
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Prerequisites
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The prerequisite for this course is a basic knowledge of cell biology, genetics, and the optical principles of microscopy, complemented by practical laboratory skills.
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Assessment methods and criteria
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Oral exam
Active participation in lectures and successful completion of an oral colloquium.
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Recommended literature
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Darzynkiewicz, Z., Crissman, H.A. (1990). Methods In Cell Biology: Flow Cytometry. San Diego.
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DARZYNKIEWICZ, Z., CRISSMAN, H.A., ROBINSON, J.P. (2000). Methods in Cell Biology: Cytometry, 3rd Edition. San Diego.
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DOLEŽEL, J., GREILHUBER, J., SUDA, J. (2007). Cytometry with Plant Cells. Wiley-VCH Verlag GmbH & Co. KGaA.
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Eckschlager, T. a kolektiv. (1999). Průtoková cytometrie v klinické praxi. Praha.
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Robinson, J.P., Darzynkiewicz, Z. (1997). Current Protocols in Cytometry. New York.
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Shapiro, H.M. (2003). Practical Flow Cytometry, 4th Edition. John Wiley and Sons, Inc., New York.
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