Flow Cytometry

Flow cytometry basics

In the technique of flow cytometry a population of cells or other particles is passed in single file in a focused stream of flowing fluid through a laser illumination source. Information about the cell or particle may be deduced from the scattered laser light in the direction of the laser path (Forward Scatter) and at ninety degrees to the incident laser light (Side Scatter). Additional information about cell phenotype and function may be gathered by an analysis of light emitted at specific frequencies from excited fluorochromes added to the cells. The flow cytometer provides a platform for the rapid collection of data generated in this way, and this information can be collated into readily decipherable graphical representations.

Flow cytometry is commonly used to investigate:

• Cell immunophenotype using fluorochrome labelled monoclonal antibodies
• The cell cycle
• Cell proliferation
• Cell apoptosis / cell death and viability
• Functional cell assays such as Calcium Release, Phagocytosis, Reactive Oxygen Species Formation and Cytoplasmic Enzyme Reactions
• Intracellular protein phosphorylation determination in cell signalling networks
• Multiple cytokine determination in a single small volume sample
• Cytoplasmic and nuclear protein determination following permeabilization of the cells
• Detection of rare cell populations such as stem cells, progenitors and immunospecific reactive cells

Equipment and facilities

Flow Cytometers

The School of Medicine has a multi-user benchtop flow cytometer located within the School within The Immunoassay Biomarker Core Laboratory, L6/094.

The Becton Dickinson LSRFortessa is a benchtop analyser equipped with three lasers, a 488nm (blue), a 633nm (red) and a 405nm (violet), which allows up to thirteen parameter analysis (two scatter detectors and eleven colour specific detectors).

In addition to the software specific to these machines, registered users have access to the analysis package Flowjo on a standalone analysis platform.

Time on the flow cytometer and Flowjo may be booked by registered staff and students using our online booking system.

Theory and practice

Excellent multimedia presentations on the theory and practice of flow cytometry:

• Introduction to Flow Cytometry Basics | Bio-Rad
• Fluorescence Theory Tutorials

A comprehensive text on the chemistry and use of fluorescent probes:

• The Handbook—A Guide to Fluorescent Probes and Labeling Technologies

Resources

Spectral Viewers

• Fluorescence SpectraViewer
• BD® Spectrum Viewer | Spectral Analyzer