Analysis of peripheral blood mononuclear cells in rodents by three-colour flow cytometry using a small-volume lysed whole blood technique.

Techniques were adapted to permit phenotypic analysis of peripheral blood leucocytes in rats and mice using small volumes of lysed whole blood and single, dual- or triple-colour immunofluorescence staining with flow cytometry. Red cell lysis using proprietary lysing solutions provides populations of lymphocytes, monocytes and granulocytes clearly differentiable by their light scatter properties. In the present study, measurement of functional and activation T lymphocyte subsets was performed by single- and dual-colour flow cytometry in both species and in excess of 5000 lymphocytes could be counted using as little as 10 microliters of whole blood. Triple-colour fluorescence, using antibodies labelled with fluorescein, phycoerythrin and biotin permitted simultaneous detection of three phenotypic markers. This novel technique has two major advantages, maximising the phenotypic information which may be obtained and using volumes of blood which are sufficiently small that they can be drawn regularly from the same animal.