PowerPoint Presentation by gioAqGh


									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
• Allows Detection Of Secreted Factors By
• Allows For Detection Of DNA Content
Principles Of Flow Cytometry
How Is It Done:

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
FSC vs SSC Dot Plot
Fluorescence And Antibodies
      To The Rescue
 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

                       E= h f
Excitation Spectra Of
Emission Spectra
Architecture Of A FacsCalibur
Analyzed Data
Unstained   ISOTYPES
GM-CSF + IL-4 (6 Days Old)

Fig. 2 GM-CSF+IL-4    GM-CSF+IL-4+PGE2
        GMF: 168.92    GMF: 1.85

        25     75      99       1

        GMF: 11.82     GMF: 391.83

        80                          99
                20     1

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