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

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) http://www.hmds.org.uk/cytometry.shtml

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 http://www.hmds.org.uk/cytometry.shtml http://www.bio.umass.edu/immunology/facs542/facsprin.htm

Procedure (cont.) Gating Sorting http://www.hmds.org.uk/cytometry.shtml 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. Enzyme-Linked Immunosorbent Assay