Flow Cytometry (FCM)

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Flow Cytometry (FCM)

• Yun-Ju Lee
• September 13, 2002

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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
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Procedure

• Disperse cells
• Tag cells w/ fluorochrome
• Antibody
• Glycoprotein receptor
• Intracellular receptor
• Add sheath fluid
• Laminar flow
• Form thread of cells
• Up to 1000s 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/facspr
in.htm
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Procedure (cont.)
http//www.hmds.org.uk/cytometry.shtml

• Gating
• Disgard data from debris, aggregates, and other
  trash
• Size (FC), viability (FC/SC ratio, fluorescent
  DNA stain)
• Sorting
• Microfluidic valves (100s cells/sec)
• Charged droplets (1000s 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.
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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