The light scatter (SS versus FS) defines three distinct populations these are the granulocytes, monocytes and lymphocytes, labelled G, M and L. Data fileįigure 4.1 shows two dot plots from some four parameter data derived from human peripheral blood leucocytes. The cells were labelled with anti-CD4-FITC and anti-CD8-PE, both these proteins are expressed on T lymphocytes. Light scatter plot The clusters labelled G, M & L arise from granulocytes, When a region is used to limit the cells that are drawn on a plot, it is termed a gate.įigure 4.1. Data from human peripheral blood leucocytes. Regions are shapes that are drawn around a population of interest on a one- or two-parameter plot. We have to adopt a different strategy we use what are called ‘regions’ and ‘gates’. However, it is impossible to visualise the correlations in multiparameter data, perhaps consisting of as many as 12 fluorescences measured on each cell. We can show the correlation between two parameters using a bivariate histogram, or cytogram, in the form of a dot, contour or density plot ( Figure 1.2). To display data from a single parameter, we can use a univariate histogram ( Figure 1.1). Whatever the program used, the principles of data analysis are the same. There are a variety of programs supplied for this purpose some of them are sold commercially, others are free. It is convenient to have a program for analysis of data files on computers in other locations. The computer program can then be used to analyse data subsequent to its acquisition off-line analysis is useful for the preparation of illustrations for publications, lecture slides, etc. If the flow cytometer can sort cells, the computer controls the sorting process.Īs data are acquired, they written to the hard drive to create a file of data, often referred to as ‘listed data’. draw regions and set gates (see below) to be used during data acquisition.select histograms and cytograms for display.adjust the settings for colour compensation (see Chapter 5.2).select and adjust the threshold (discriminator) settings (see Chapter 2.5.1).
select logarithmic or linear amplification.adjust the gain settings on the amplifiers.select area, width or height on different parameters (for pulse processing, see Chapter 2.5.2).The computer program controls the cytometer during data acquisition. All flow cytometers have a computer associated with them.
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