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Flow Cytometry - PowerPoint PPT Presentation


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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 is a powerful technique for characterizing immune cells
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
how is it done
How Is It Done:

http://biology.berkeley.edu/crl/flow_cytometry_basic.html

limitations with light scattering
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
fluorescent dyes and antibodies
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)
principles of fluorescence
Principles Of Fluorescence

E= h f

 = f 

slide14

Unstained

ISOTYPES

slide16

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