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Understanding Cell Viability: Techniques and Applications in Flow Cytometry

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The goal of cell viability studies is to differentiate live cells from dead and apoptotic cells, allowing for the calculation of the percentage of viable cells in experiments. Methods such as Trypan blue staining and flow cytometry provide insights into cell health. Flow cytometry, with its components – fluidics, optics, and lasers – processes large volumes of data efficiently. By analyzing parameters like forward scatter (FSC) and side scatter (SSC), researchers can determine cell size, granularity, and further isolate populations for detailed study.

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Understanding Cell Viability: Techniques and Applications in Flow Cytometry

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Presentation Transcript

  1. Cell viability studies Sepideh Khoshnevis

  2. The Goal • To distinguish live cells from dead and apoptotic cells in order to calculate the the percentage of viable cells for each experiment

  3. Cell viability study • Trypan blue studies • Finding data for a small population • a good first order approximation • Flow cytometry • Processing a large volume of data in a reasonable time

  4. BD FACSCalibur

  5. How does flow cytometer work? • It is made up of three systems • Fluidics • Optics • Laser • Filters • electronics

  6. introduction to flow cytometry, BD biosciences

  7. fluidics • Hydrodynamic focusing introduction to flow cytometry, BD biosciences

  8. Light Scatter • Forward scatter (FSC) • Proportional to cell size • Side scatter (SSC) • Proportional to cell granularity or internal complexity • FSC vs. SSC plot • Isolating mixture of cell population

  9. Differentiation of cell types introduction to flow cytometry, BD biosciences

  10. Fluorescence • Fluorescent dyes • Fluorochrome conjugated antibodies

  11. Optical bench diagram introduction to flow cytometry, BD biosciences

  12. Signal detection • When the particles enters the laser beam light signal is being produced • Light is converted to voltage by photodetectors • Photodiode • Photomultiplier tube • Size of voltage pulse depends on the number of photons detected • The height of the voltage pulse dictate what channel it falls under

  13. Creation of voltage pulse

  14. Data display • Histogram • Dot plots • Multi dimensional plots

  15. Current protocols in cytometry

  16. Current protocols in cytometry

  17. Current protocols in cytometry

  18. Gating • Isolating the population of interest • Cluster analysis • Further analysis of a subpopulation

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