Flow Cytometry

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Flow Cytometry

• Basic Principles, Instrumentation, and Practices

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Introduction/Basic Facts

• Laser-optics-computer based technology for
  measuring the characteristics of biological
  particles
• Bioparticles can be whole cells or prepared
  cellular constituents
• Uses a one particle at a time approach for
  analysis rather than measuring a bulk property
• Measures the light scattering and fluorescence
  properties of particles

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Biological Cell
5-20 µm in diameter
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Cell Labeling Techniques

• Antibodies Conjugated to Fluorochromes
• FITC, Phycoerythrin, proprietary
• Cytoplasmic Dyes/Stains
• Permeant, nonpermeant
• Nuclear stains
• Membrane dyes

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Most Common Cell Labeling
Single Antibody
Dual Antibody
Internal
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Flow Cytometry Applications

• Detection of Intracellular Cytokine Production
• Detection of Intracellular/intranuclear antigens
• Estimation of cell viability
• Cell transmembrane potential measurements
• Measurement of oxidative metabolism
• Measurement of environmental particulate uptake
• Detection of intracellular cyclins

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List of Flow Cytometry Applications

• Pharmacokinetic monitoring
• Quantitation of proteins inserted into membranes
• Quantitation of electropermeabilization
• Cell cycle analysis
• Analysis of apoptosis
• Cell Sorting
• Chromosome sorting
• Up to 7 fluorochrome analysis

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• 1969 Los Almos Labs created the first flow
  cytometer
• Normally have a dedicated operator
• USF researchers have access to the Moffitt Core
  Flow Cytometry Facility

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Flow Cytometer Block Diagram
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Spectral Overlap
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Compensating forSpectral Overlap
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Control Samples

• Compensation for 2 or more colors
• Negative
• Unlabeled cell zero reference point
• Isotype control nonspecific binding
• Positive make sure that labeled antibody is
  functional

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Examples of Flow Cytometric Analysis

• One parameter FSC analysis for cell size
• One parameter FL1 analysis for drug uptake
• Two parameter fluorescence analysis for dual
  labeled cells
• Cell Sorting

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One Parameter FSC Analysis
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One Parameter FL1 Analysis
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One Parameter FL1 Analysis
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Fluorescence Two Color Analysis
Unfused
Fused
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Fluorescence Two Color Analysis
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Cell Sorting
Filters/ detectors
/- V
/- V