Download
flow cytometry n.
Skip this Video
Loading SlideShow in 5 Seconds..
Flow Cytometry PowerPoint Presentation
Download Presentation
Flow Cytometry

Flow Cytometry

437 Views Download Presentation
Download Presentation

Flow Cytometry

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  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