Basic Principles, Instrumentation, and Practices 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 FL1 analysis for drug uptake Two parameter fluorescence analysis for dual labeled cells Cell Sorting

One Parameter FSC Analysis

One Parameter FL1 Analysis

One Parameter FL1 Analysis

Fluorescence Two Color Analysis Unfused Fused

Fluorescence Two Color Analysis

Cell Sorting Filters/ detectors +/- V +/- V