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Flow cytometry stands as a crucial method in biological research with applications spanning across various
disciplines, including microbiology, ecology, immunology, and clinical diagnostics. As a high-throughput single-cell method, it allows for the analysis of millions of cells with more than 30 parameters. It is also fundamental for sorting single cells for downstream analysis of defined cell subsets for single-cell RNAseq, etc. To be able to leverage flow cytometry to its full potential, an understating of the technology is necessary. During Basics of Flow cytometry, students will attend theoretical lectures with follow-up practical hands-on sections that will cover all essentials of flow cytometry. During the hands-on sessions held at the flow cytometry core facility of the Institute of Hematology and Blood Transfusion, students will have hands-on experience with advanced classical and spectral flow cytometers. They will be able to learn instrument setup. We plan to collaborate with flow cytometer manufacturers to also demo interesting instruments, that are not available in the core facility. Last update: Marková Hana, RNDr., Ph.D. (18.04.2024)
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Credits are awarded for participation in the practical part of the course (a max. of 2 absences is allowed). An oral exam is required to pass the course. The questions cover the learning material from the presentations and the practical part of the course. Last update: Musil Jan, RNDr., Ph.D. (13.02.2025)
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Topic overview: 1. How does a flow cytometer work - In this section, we will discuss how a cytometer works and describe the function of individual components: a. Optical system - We will discuss the individual components of excitation optics, i.e., laser types, mirrors, and collection optics, i.e., dichroic mirrors, and methods of wave-length demultiplexing. We will compare classical and spectral cytometers. b. Fluidics - Here, we will discuss fluidics, hydrodynamic and acoustic focusing, and flow rate. c. Electronics - Students will learn about light detection using different photon detectors. How the signal is processed, i.e., measurements of pulse Area, Hight and Width and their use. They will also learn how data is visualized, i.e., dot plots, contour plots, pseudocolor plots, and histograms. When and how to use individual types of plots. Students will learn about system characteristics such as sources of noise and detector linearity and how to define them and use them for proper cytometer setup. This includes a system setup for complex and autofluorescent samples, and tips on how to ensure we see what we want to see.
2. Fluorochromes and fluorescence a. Fluorochromes and fluorescence - Students will learn what fluorochromes are and the basic physics behind fluorescence. b. Fluorochrome characteristics and how to evaluate them - Students will learn about excitation and emission spectra and how they impact emission detection. What is fluorescence brightness, and how to quantify it. We will discuss spectral spill-over spreading and its impact on panelresolution. The sources of spreading. How to measure spreading and quantify it on classical and spectral cytometer. c. Compensation and Unmixing - In this section, we will discuss what are compensation and unmixing and why are they needed. We will explore the relation between compensation and spreading. The student will learn how to perform manual and automatic compensation, spectral unmixing, and how to evaluate their performance.
3. Panel design principles and planning a flow cytometry experiment - In this section, we will summarize the previous knowledge and show a basic workflow of panel design and principles of panel optimization. We will discuss antibody titration and how to perform it. Students will learn how to evaluate panel performance. Furthermore, we will show how to plan an experiment and which controls are needed, i.e., compensation controls, fluorescence-minus one (FMO), isotype controls, and biological controls. We will discuss cytometer standardization. We will explain basic principles of staining such as staining buffer composition, blocking and usage of fixatives. Effects of temperature on staining
4. Basic principles of data analysis and interpretation a. Data quality control b. Compensation/unmixing quality control c. Data transformation d. Understanding high-parameter data and how to spot artifacts. e. Principles of gating, data visualization, exporting of statistics f. An immunologist tool-box for advanced analysis without programming skills
5. Cell sorting - In this section we will explain how a cell sorter works, what are the difference between a cell sorter and an analyzer. We will explain how to prepare a sample and show how to setup a simple cell sort.
Last update: Marková Hana, RNDr., Ph.D. (18.04.2024)
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