Download - Flow cytometry
Flow Cytometry
• 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
Flow Cytometry Is A Powerful Technique For Characterizing Immune Cells
Principles Of Flow Cytometry
http://biology.berkeley.edu/crl/flow_cytometry_basic.html
How Is It Done:
• 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
Limitations With Light Scattering
FSC vs SSC Dot Plot
Fluorescence And Antibodies To The Rescue
• 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)
Fluorescent Dyes And Antibodies
Principles Of Fluorescence
E= h f
= f
Excitation Spectra Of Fluorochromes
Emission Spectra
Architecture Of A FacsCalibur Instrument
Analyzed Data
ISOTYPESUnstained
GM-CSF
GM-CSF + IL-4 (6 Days Old)
2080
GM-CSF+IL-4+PGE2
199
991
GM-CSF+IL-4
7525
GMF: 11.82
GMF: 168.92
GMF: 391.83
GMF: 1.85
Fig. 2