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