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).
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-
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.
Figure 1.4. A histogram showing fluorescence against cell number. The fluorescence signals have been digitised into 1024 channels. The y-
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.