Flow Cytometry Basic Principles, Instrumentation, and Practices.
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Transcript of Flow Cytometry Basic Principles, Instrumentation, and Practices.
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Flow Cytometry
Basic Principles, Instrumentation, and Practices
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Introduction/Basic Facts
• Laser-optics-computer based technology for Laser-optics-computer based technology for measuring the characteristics of biological measuring the characteristics of biological particlesparticles
• Bioparticles can be whole cells or prepared Bioparticles can be whole cells or prepared cellular constituentscellular constituents
• Uses a one particle at a time approach for Uses a one particle at a time approach for analysis rather than measuring a bulk propertyanalysis rather than measuring a bulk property
• Measures the light scattering and fluorescence Measures the light scattering and fluorescence properties of particlesproperties of particles
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Biological Cell
5-20 µm in diameter5-20 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 Single AntibodyAntibody
Dual Dual AntibodyAntibody InternalInternal
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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 OverlapSpectral Overlap
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Compensating for
Spectral 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 sizeOne parameter FSC analysis for cell size
• One parameter FL1 analysis for drug uptakeOne parameter FL1 analysis for drug uptake
• Two parameter fluorescence analysis for dual Two parameter fluorescence analysis for dual labeled cells labeled cells
• Cell SortingCell Sorting
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One Parameter FSC Analysis
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One Parameter FL1 AnalysisOne Parameter FL1 Analysis
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One Parameter FL1 AnalysisOne Parameter FL1 Analysis
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Fluorescence Two Color AnalysisFluorescence Two Color Analysis
UnfusedUnfused FusedFused
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Fluorescence Two Color Analysis
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Cell Sorting
Filters/ Filters/ detectorsdetectors
+/- V+/- V +/- V+/- V