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