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Flow Cytometry

Flow Cytometry

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Flow Cytometry

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Presentation Transcript

  1. Flow Cytometry Basic Principles, Instrumentation, and Practices

  2. Introduction/Basic Facts • Laser-optics-computer based technology for measuring the characteristics of biological particles • Bioparticles can be whole cells or prepared cellular constituents • Uses a one particle at a time approach for analysis rather than measuring a bulk property • Measures the light scattering and fluorescence properties of particles

  3. Biological Cell 5-20 µm in diameter

  4. Cell Labeling Techniques • Antibodies Conjugated to Fluorochromes • FITC, Phycoerythrin, proprietary • Cytoplasmic Dyes/Stains • Permeant, nonpermeant • Nuclear stains • Membrane dyes

  5. Most Common Cell Labeling Single Antibody Dual Antibody Internal

  6. Flow Cytometry Applications • Detection of Intracellular Cytokine Production • Detection of Intracellular/intranuclear antigens • Estimation of cell viability • Cell transmembrane potential measurements • Measurement of oxidative metabolism • Measurement of environmental particulate uptake • Detection of intracellular cyclins

  7. List of Flow Cytometry Applications • Pharmacokinetic monitoring • Quantitation of proteins inserted into membranes • Quantitation of electropermeabilization • Cell cycle analysis • Analysis of apoptosis • Cell Sorting • Chromosome sorting • Up to 7 fluorochrome analysis

  8. 1969 Los Almos Labs created the first flow cytometer • Normally have a dedicated operator • USF researchers have access to the Moffitt Core Flow Cytometry Facility

  9. Flow Cytometer Block Diagram

  10. Spectral Overlap

  11. Compensating forSpectral Overlap

  12. Control Samples • Compensation – for 2 or more colors • Negative • Unlabeled cell – zero reference point • Isotype control – nonspecific binding • Positive – make sure that labeled antibody is functional

  13. Examples of Flow Cytometric Analysis • One parameter FSC analysis for cell size • One parameter FL1 analysis for drug uptake • Two parameter fluorescence analysis for dual labeled cells • Cell Sorting

  14. One Parameter FSC Analysis

  15. One Parameter FL1 Analysis

  16. One Parameter FL1 Analysis

  17. Fluorescence Two Color Analysis Unfused Fused

  18. Fluorescence Two Color Analysis

  19. Cell Sorting Filters/ detectors +/- V +/- V