1. Flow Cytometry (FCM)
Yun-Ju Lee
September 13, 2002

2. Introduction “Flow” + “Cell” + “Measurement” = FCM (FACS®)
What it does:
Measures cell functionalities and quantities (Fluorescence)
Measures cell sizes (scattering)
Sort different cells (very selective)
Applications
Study cellular mechanisms
Viral binding
Gene expression
Immune recognition
Clinical studies
Cancer cell determination
WBC ID (eg CD-4)

3. Procedure Disperse cells Tag cells w/ fluorochrome Add sheath fluid
Antibody
Glycoprotein receptor
Intracellular receptor
Add sheath fluid
Laminar flow
Form “thread” of cells
Up to 1000’s cells/sec
Excite with laser
Detection:
Fluorescence (photodiode array
Light scattering

4. Procedure (cont.) Gating Sorting
Procedure (cont.)
Gating
Disgard data from debris, aggregates, and other trash
Size (FC), viability (FC/SC ratio, fluorescent DNA stain)
Sorting
Microfluidic valves (100’s cells/sec)
Charged droplets (1000’s cells/sec)
Flow cytometry data analysis. Left-hand plot show a one-colour histogram plot of CD8 expression by peripheral blood lymphocytes. Approximately 38% of events fall between the marker boundaries, and are therefore regarded as CD8 +ve. The centre plot also shows CD8 expression on PB lymphocytes, but depicts the relationship between CD8 and the T-cell marker CD3. The right-hand plot shows a population of CD34 +ve 'stem cells' plotted against side scatter.

5. FCM vs. other techniques
Great for heterogeneous populations
Samples, analyzes, and sorts all subpopulations
If homogeneous (e.g. only looking for conc of 1 antibody type)  other techniques (ELISA, RIA)
Sensitive but slow sorting
Slow throughput/yield loss
106 cells/hour (1 mouse  108 lymphocytes)
Alternatives: affinity columns, density gradient, antibody panning, etc.
Enzyme-Linked Immunosorbent Assay