The setup of a sort depends entirely on which cells or particles are needed.
As already noted, it is possible to sort according to antigen expression, fluorescent
protein expression, nucleic acid content, or functionality. In all cases, a preliminary
experiment on an analytical cytometer is always worthwhile. In addition,
in most cases, a dye that will specifically identify dead cells should be added in
order to exclude them from the sort. Although these cells may not be problematic
when cells are to be recultured, it would be important to exclude them if specific
numbers of cells were needed for functional studies or if cells were to be used to
analyze protein expression or mRNA content. Examples of viability dyes include
propidium iodide (used at 5 Rg/mL), 7-aminoactinomycin-D (2 Rg/mL), TO-PRO-3
(200 nM), and DAPI (4e, 6-Diamidino-2-phenylindole; 200 ng/mL). The dye
used will depend on which combination of fluorochromes will be used in the
sort to identify the populations of interest.