Flow Cytometry Basic Principles Instrumentation and Practices IntroductionBasic
Flow Cytometry Basic Principles, Instrumentation, and Practices
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
Biological Cell 5 -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 Antibody Dual Antibody Internal
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 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 size • One parameter FL 1 analysis for drug uptake • Two parameter fluorescence analysis for dual labeled cells • Cell Sorting
One Parameter FSC Analysis
One Parameter FL 1 Analysis
One Parameter FL 1 Analysis
Fluorescence Two Color Analysis Unfused Fused
Fluorescence Two Color Analysis
Cell Sorting Filters/ detectors +/- V
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