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Flow Cytometry
Basic Principles, Instrumentation, and Practices
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
Biological Cell
5-20 µm in diameter5-20 µm in diameter
Cell Labeling Techniques
• Antibodies Conjugated to Fluorochromes– FITC, Phycoerythrin, proprietary
• Cytoplasmic Dyes/Stains– Permeant, nonpermeant
• Nuclear stains
• Membrane dyes
Most Common Cell Labeling
Single Single AntibodyAntibody
Dual Dual AntibodyAntibody InternalInternal
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
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
• 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
Flow Cytometer Block Diagram
Spectral OverlapSpectral Overlap
Compensating for
Spectral Overlap
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
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
One Parameter FSC Analysis
One Parameter FL1 AnalysisOne Parameter FL1 Analysis
One Parameter FL1 AnalysisOne Parameter FL1 Analysis
Fluorescence Two Color AnalysisFluorescence Two Color Analysis
UnfusedUnfused FusedFused
Fluorescence Two Color Analysis
Cell Sorting
Filters/ Filters/ detectorsdetectors
+/- V+/- V +/- V+/- V