A subset of the enormous number of diverse applications to which the flow cytometric technique lends itself has been explored. Flow cytometry and cell sorting has been used in immunology, for example in the definition of multiple cellular subpopulations in the developing thymus, in parasitology, where the presence of malaria parasites in red blood cells has been measured, in haematology where progenitor cells of various lineages have been sorted from a range of haemopoietic source tissues, and in other cell biological or clinical research either where a defined subgroup of cells needs to be sorted to high purity from a cell sample, for example, pure neutrophils from mouse bone marrow, or where discrete subpopulations, perhaps those associated with a disease state, are to be enumerated.
Early thymus studies have sought to monitor cell differentiation in organ cultures of whole foetal thymuses by light scatter alone. Subsequent work has concentrated on the blinding patterns of a panel of fluorescent monoclonal antibodies and lectins, for example, Ly-1, Ly-2, L3T4, Thy-1, H-2 and PNA and then these in combination with cytotoxicity to remove major cell groups using, for example, 2A2 and finally, those methods together with a topological staining technique using either Hoechst 33258 or a bromobimane intracellular stain to relate phenotype to position.
The staining procedures first developed for characterising viable plasmodium berghei infection of mouse red blood cells using Hoechst 33258 have been extended to human malaria (p. falciparum) and further, to allow a routine parasitaemia assay on fixed infected cells where a linear response and very low false positive cell rate (<0.1%) have been recorded.
In sorting haemopoietic progenitors, the criteria used, apart from binding of various specific monoclonal antibodies have included the binding of fluorescinated lectins, for example PWM for mouse cells and FBP for human cells, the forward scattering of laser light or the scattering at high angle and the emission of blue, ultra-violet-excited autofluorescence. The latter three parameters have been used in other work for the sorting of neutrophils to high purity from mouse peritoneum or bone marrow and, further, the blue autofluorescence has been shown to be an indication of granulocyte activation.