Triple labeling with two-color immunofluorescence using one light source: a useful approach for the analysis of cells positive for one label and negative for the other two

Many laboratories do not have access to a flow cytometer allowing three-color immunofluorescence analysis through the use of multiple light sources. In view of the usefulness of such analyses in the dissection of cell parameters, we describe an approach permitting the study of three labels by using one light source and the two-color immunofluorescence assay. It is useful for the enumeration of cell subpopulations positive for one label and negative for two or more others as well as for qualitative analysis concerning the expression of these labels. This approach is simple and rapid; it does not require additional material and technical steps other than that used in the two-color immunofluorescence assay. Briefly, it consists of the use of a label coupled to a dye (PE or FITC or instance) and two different labels coupled to the other dye. An argon ion laser, operating at 488 nm and 60 mW, excites both fluorescein and phycoerythrin conjugated antibodies. We provided a general example, using three hypothetical labels (X, Y, and Z), and four practical applications: CD3+CD4CD8- and CD8+CD16-CD3- peripheral blood lymphocytes, CD2+CD16-CD3- and CD56+CD16-CD3- peripheral blood, and decidual infiltrating lymphocytes.