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-
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.
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: