Flow Cytometry
<ul><li>Allows For Detection Of Surface Markers Of Cells </li></ul><ul><li>Allows For Detection Of Intracellular Factors <...
Principles Of Flow Cytometry
How Is It Done:  http://biology.berkeley.edu/crl/flow_cytometry_basic.html
<ul><li>Some Information Can Be Obtained </li></ul><ul><li>FSC Correlates With Cell Size </li></ul><ul><li>SSC Correlates ...
FSC vs SSC Dot Plot
Fluorescence And Antibodies  To The Rescue
<ul><li>Fluorochromes Are Molecules That Emit Fluorescence Upon Excitation With Light  </li></ul><ul><ul><li>Ex. FITC (Flu...
Principles Of Fluorescence E= h f     = f  
Excitation Spectra Of Fluorochromes
Emission Spectra
Architecture Of A FacsCalibur Instrument
Analyzed Data
ISOTYPES Unstained
GM-CSF GM-CSF + IL-4  (6 Days Old)
20 80 GM-CSF+IL-4+PGE 2 1 99 99 1 GM-CSF+IL-4 75 25 GMF: 11.82 GMF: 168.92 GMF: 391.83 GMF: 1.85 Fig. 2
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Flow cytometry

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Flow cytometry

  1. 1. Flow Cytometry
  2. 2. <ul><li>Allows For Detection Of Surface Markers Of Cells </li></ul><ul><li>Allows For Detection Of Intracellular Factors </li></ul><ul><li>Allows Detection Of Secreted Factors By Cells </li></ul><ul><li>Allows For Detection Of DNA Content </li></ul>Flow Cytometry Is A Powerful Technique For Characterizing Immune Cells
  3. 3. Principles Of Flow Cytometry
  4. 4. How Is It Done: http://biology.berkeley.edu/crl/flow_cytometry_basic.html
  5. 5. <ul><li>Some Information Can Be Obtained </li></ul><ul><li>FSC Correlates With Cell Size </li></ul><ul><li>SSC Correlates With Internal Complexity </li></ul><ul><li>To Distinguish Between 2 Cell types </li></ul><ul><ul><li>A. Size Has To Be Different OR </li></ul></ul><ul><ul><li>B. Internal Complexity i.e amount of granules </li></ul></ul><ul><li>If These Two Parameters Are The Same, Then No Distinction Can Be Made </li></ul><ul><li>See The Following Figure </li></ul>Limitations With Light Scattering
  6. 6. FSC vs SSC Dot Plot
  7. 7. Fluorescence And Antibodies To The Rescue
  8. 8. <ul><li>Fluorochromes Are Molecules That Emit Fluorescence Upon Excitation With Light </li></ul><ul><ul><li>Ex. FITC (Fluorescein Isothiocyanate) </li></ul></ul><ul><ul><li>PE (Phycoerythrin) </li></ul></ul><ul><ul><li>PerCP (Peridinin Chlorophyll Protein) </li></ul></ul><ul><ul><li>APC (Allophycocyanin) </li></ul></ul><ul><li>Some Fluorochromes Are Proteins, Some Are Small Organic Compounds </li></ul><ul><ul><li>Ex. PE (Phycoerythrin)-Protein </li></ul></ul><ul><ul><li>Ex. FITC (Fluorescein Isothiocyanate) </li></ul></ul>Fluorescent Dyes And Antibodies
  9. 9. Principles Of Fluorescence E= h f  = f 
  10. 10. Excitation Spectra Of Fluorochromes
  11. 11. Emission Spectra
  12. 12. Architecture Of A FacsCalibur Instrument
  13. 13. Analyzed Data
  14. 14. ISOTYPES Unstained
  15. 15. GM-CSF GM-CSF + IL-4 (6 Days Old)
  16. 16. 20 80 GM-CSF+IL-4+PGE 2 1 99 99 1 GM-CSF+IL-4 75 25 GMF: 11.82 GMF: 168.92 GMF: 391.83 GMF: 1.85 Fig. 2

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