flow cytometry basic principles, instrumentation, and practices

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