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

Data display

You will need to be familiar with the different types of data display used in flow cytometry.

To look at a single parameter, a univariate histogram is used; cell number is displayed against intensity of the measured parameter (Figure 1.4).









To observe two parameters, a correlated dot plot is used; each dot on plot represents a single cell and shows the values of the two parameters measured (Figure 1.5). A two parameter plot is often referred to as a cytogram.














Univaiate histogram and cytograms are displayed on the computer and updated during data acquisition.

Contoured cytograms and density plots are sometimes used to display two parameter data (figure 1.6).









 


 


Figure 1.6. A density plot  (A) and a contour plot (B) of the scattered light from human peripheral blood leucocytes (the identical data to that in Figure 1.5).

Safety

Flow cytometric work usual involves handling biological cells. Normal laboratory safety procedures should be followed.

Some of the chemicals used for staining cells (particularly the DNA-binding dyes, such as propidium iodide and ethidium bromide) are putative carcinogens and/or mutagens. They should be handled using the appropriate laboratory procedure, including the use of protective gloves.

Bench top cytometers are fully enclosed. The biological hazards associated with them relate to sample preparation rather than the instrument itself. Any possible safety risk can be minimised by fixing samples before analysis, whenever the experimental protocol permits. After running unfixed samples, the sample lines should be decontaminated by running a disinfectant, such as dilute bleach. The contents of the waste container should be sterilised by the addition of disinfectant before disposal in compliance with your institution's guidelines.

There are particular hazards associated with instruments that sort cells electrostatically by charged droplet formation. These will be discussed in relation to cell sorting in Unit 2.

Continued


Figure 1.4. A histogram showing fluorescence against cell number. The fluorescence signals have been digitised into 1024 channels. The y-axis shows the number of cells in each channel (events). The histogram shows cells whose DNA has been labelled with a fluorescence dye. The intensity of fluorescence is a measure of the DNA content of the cells.


Figure 1.5. A correlated dot plot (cytogram) showing the scattered light from human peripheral blood leucocytes. Each dot represents one cell. The circled dot has the intensity of the forward scattered light (FS) and the light scattered at right angles (RS) marked.