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Flow Cytometry

Flow Cytometry. Flow Cytometry Is A Powerful Technique For Characterizing Immune Cells. Allows For Detection Of Surface Markers Of Cells Allows For Detection Of Intracellular Factors Allows Detection Of Secreted Factors By Cells Allows For Detection Of DNA Content.

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Flow Cytometry

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  1. Flow Cytometry

  2. Flow Cytometry Is A Powerful Technique For Characterizing Immune Cells • Allows For Detection Of Surface Markers Of Cells • Allows For Detection Of Intracellular Factors • Allows Detection Of Secreted Factors By Cells • Allows For Detection Of DNA Content

  3. Principles Of Flow Cytometry

  4. How Is It Done: http://biology.berkeley.edu/crl/flow_cytometry_basic.html

  5. Limitations With Light Scattering • Some Information Can Be Obtained • FSC Correlates With Cell Size • SSC Correlates With Internal Complexity • To Distinguish Between 2 Cell types • A. Size Has To Be Different OR • B. Internal Complexity i.e amount of granules • If These Two Parameters Are The Same, Then No Distinction Can Be Made • See The Following Figure

  6. FSC vs SSC Dot Plot

  7. Fluorescence And Antibodies To The Rescue

  8. Fluorescent Dyes And Antibodies • Fluorochromes Are Molecules That Emit Fluorescence Upon Excitation With Light • Ex. FITC (Fluorescein Isothiocyanate) • PE (Phycoerythrin) • PerCP (Peridinin Chlorophyll Protein) • APC (Allophycocyanin) • Some Fluorochromes Are Proteins, Some Are Small Organic Compounds • Ex. PE (Phycoerythrin)-Protein • Ex. FITC (Fluorescein Isothiocyanate)

  9. Principles Of Fluorescence E= h f  = f 

  10. Excitation Spectra Of Fluorochromes

  11. Emission Spectra

  12. Architecture Of A FacsCalibur Instrument

  13. Analyzed Data

  14. Unstained ISOTYPES

  15. GM-CSF + IL-4 (6 Days Old) GM-CSF

  16. GM-CSF+IL-4 GM-CSF+IL-4+PGE2 Fig. 2 GMF: 1.85 GMF: 168.92 25 75 99 1 GMF: 11.82 GMF: 391.83 99 80 1 20

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