presentasi metode flow cytometry (PArt, types, aplication)
- 1. Flow Cytometry Understanding Principles and Applications
- 2. Introduction Flow cytometry is a laser-based technology for analyzing and measuring physical and chemical characteristics of cells or particles in fluid. This presentation covers its principles, instrumentation, applications, limitations, and future perspectives.
- 3. Table of contents - Definition of Flow Cytometry - Principles of Flow Cytometry - Instrumentation/Parts of Flow Cytometry - Light Scattering and Fluorescence - Protocol for Flow Cytometry - Types of Flow Cytometry - Applications of Flow Cytometry - Limitations of Flow Cytometry
- 4. Definition of Flow Cytometry This technology is widely used in clinical and research settings for various applications such as immunology, cell biology, and cancer research. Flow cytometry employs lasers to illuminate cells as they flow in a stream and uses detectors to capture the resulting light signals. The technique allows for the simultaneous analysis of multiple parameters at a high throughput, enabling rapid data collection from thousands of particles per second. Fluorescence labels introduced to the cells provide specific information about cellular markers and can indicate the presence of
- 5. Principles of Flow Cytometry As particles flow through a focused laser beam in a hydrodynamically focused manner, the interaction of light with particles leads to forward scattering and side scattering, providing distinct information about size and granulation. Forward scatter indicates the overall size of particles, while side scatter is correlated with internal complexity, such as granules or organelles within cells, aiding in distinguishing between different cell types. The use of fluorescent dyes that bind to specific cellular markers, such as proteins or nucleic acids, enhances the ability to identify and
- 6. Instrumentation/Parts of Flow Cytometry The fluidics system uses a sheath fluid to focus the sample into a narrow stream, allowing individual particles to pass through the laser one at a time for precise analysis. In the optics system, the lasers are aligned to focus on the sample stream, with optical filters used to select the specific wavelengths of light that are relevant for detection. Detectors, such as photomultiplier tubes (PMTs) or avalanche photodiodes, capture scattered light and emitted fluorescence and convert these light signals into electrical signals. Advanced flow cytometers include multiple lasers and detectors to
- 7. Light Scattering and Fluorescence The degree of light scattering is influenced by the size, shape, and refractive index of the particles, allowing for detailed characterization of various cell types and their states. In flow cytometry, forward scatter and side scatter are two distinct measurements that provide complementary information about cell size and internal complexity. By utilizing multiple fluorescent markers, researchers can identify specific cell populations and track changes in cellular behavior, such as activation or apoptosis. Different fluorescent dyes have unique excitation and emission
- 8. Protocol for Flow Cytometry To achieve single-cell suspension, cell clusters must be dissociated using enzymatic digestions or mechanical methods, ensuring accurate analysis without aggregation. In antibody staining, it is essential to optimize the concentration of antibodies to prevent nonspecific binding while ensuring sufficient fluorescent signal for detection. Samples should be incubated for a specific duration at an appropriate temperature, often in the dark, to enhance the binding of antibodies to their target antigens. Blocking agents can be used in the staining protocol to reduce
- 9. Types of Flow Cytometry Traditional flow cytometers are typically characterized by a single or a few lasers and can perform basic analysis of cell populations, making them suitable for straightforward applications in research and diagnostics. Acoustic Focusing Cytometers utilize sound waves to manipulate and focus cells in the fluid stream, enhancing detection accuracy and allowing for the analysis of larger particles without the shear forces present in traditional systems. Cell Sorters are advanced types of flow cytometers specifically designed to isolate and collect specific cell populations based on
- 10. Applications of Flow Cytometry Used in biology, pathology, immunology, virology, and plant biology. Detect malignancy in bodily fluids, separate cells of interest, and analyze DNA content. Can identify different stages of cell death and the effects of treatments.
- 11. Limitations of Flow Cytometry Does not provide intracellular localization of proteins. Sample preparation can be time-consuming. High costs and requires skilled technicians.
- 12. Conclusion Flow cytometry is a critical tool in modern biology and medicine, allowing for detailed analysis of cell properties, though it faces limitations that can impact its effectiveness.
- 13. References - Biotech, M. (2018). Flow cytometry instrumentation – an overview. Current Protocols in Cytometry. - McKinnon K. M. (2018). Flow Cytometry: An Overview. Current protocols in immunology. - Dean, P.N. and Hoffman, R.A. (2007). Overview of Flow Cytometry Instrumentation.
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