What it Introduction
　　“Flow” + “Cell” + “Measurement” = FCM (FACS®)
　　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)
　　Procedure

Procedure
　　Disperse cells
　　Tag cells w/ fluorochrome
　　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

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.

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 mouse108 lymphocytes)
Alternatives: affinity columns, density gradient, antibody panning, etc.