What is flow cytometry?

The flow system

Light scatter

Fluorescence measurement

Multiparametric measurement

Advantages of flow cytometry

Disadvantages of flow cytometry

Sample preparation

Applications

Data display

Safety

Further reading

Self Assessment

Introductory Contents Further reading Units 	Introduction  The flow cytometer 	Data analysis 	Immunofluorescence 	Some clinical applications  DNA and the cell cycle Other applications Cell proliferation Cell death Assignment 1 Assignment 2

Disadvantages of flow cytometry

Sample preparation

When preparing samples for flow cytometry, the objective is to produce a suspension of single cells free of debris. Blood contains a convenient suspension of single cells; aspirates, particularly from bone marrow or effusions, often contain few clumps. Samples from solid tissues have to be treated, mechanically or with enzymes or both, to produce single cell suspensions.

When studying cultured cells, cells growing in suspension cultures present few problems. Cells growing attached to a plastic substrate have to be brought into a single cell suspension, usually by treatment with trypsin/EDTA. Care must be taken to produce as good a suspension with as few clumps as possible without over-treating with trypsin; over-treatment can lyse some of the cells.

When performing an assay for the first time, samples should be monitored using a light microscope.

Sample preparation is the key to good flow cytometry. Poorly prepared samples will give poor results.

Applications

The rapid growth in the use of flow cytometry over the last twenty years is due to three factors.

These factors are reflected in the large number of applications for the technology.

The most common application of flow cytometry is immunofluorescence analysis. Its use is widespread in both haematology and immunology. Antibodies are used to measure the expression of cell surface markers and (after the cells have been permeabilised) intracytoplasmic and nuclear proteins.

The second most common application (particularly in a research laboratory) is the measurement of DNA content, which is used to analyse the cell cycle.

Other common applications are the analysis of cell proliferation and the study of death, particularly apoptosis.

A range of functional parameters of the cell may be measured. These include:

Other biological applications include:

Continued