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 mouse 108 lymphocytes)
Alternatives: affinity columns, density gradient, antibody panning, etc.
