3.1.1 Applications of flow cytometry
How cytometry can measure the size and granularity of cells from their light scattering properties, in addition to determining the expression of antigenic markers by fluorescence detection. Flow cytometry may also be used to measure the relative uptake of fluorescent endocytic and phagocytic tracers (see Sections
5.2 and 5.3). Macrophages can be distinguished and sorted from other cell types in a complex cell mixture on the basis of size and granularity, using the forward and side scattcr parameters, and by the expression of macrophage-restricted antigens using fluorescent antibody labelling.
3.1.2 Practical considerations for detecting macrophages by flow cytometry
A few points on the analysis of macrophages by flow cytometry will be mentioned here. However, for detailed information on the techniques required see refs 7 and 8. Due to their large size, macrophages have a large forward scatter making it easy to sort them from lymphocytes in mixed cell populations. Macrophages are heterogeneous with respect to then size within a given isolated population, which results in a broad range of forward scatter values when compared with other cell types.
Double and triple labelling of macrophages for the flow cytomcter requires adjustment of the flow cytomcter to compensate for the broad emission spectra of the commonly used fluorochromes (see Tab It 2). The broad emission spectrum of the fluorochromes is responsible for the overlap into the spectrum of other fluorochromes. To help in the accuratc setting of the colour compensation, it is important that there is little cross-reactivity between reagents and the macrophages cultured in phenol red-free medium to minimize acquired autofluorescence. The colour compensation should be set up with control macrophages labelled with only one of the fluorochromes used. It is recommended that a large number of control cells are prepared since balancing compensation can require many cells. A good description on how to set up a flow cytometer for double and triple labelling can be accessed at: http://www.molbiol.ox.ac.uk/ pathology/tig/facs.html
Macrophage adherence to plastic test-tubes during incubation is a problem so use less sticky polypropylene vessels and incubate and wash the cells at 4°C. Ensure that the macrophages are in a single cell suspension as clumps can alter the population distribution on the flow cytometer.
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