MULTIPLE LABELING USING 2-COLOR IMMUNOFLUORESCENCE WITH ONLY ONE LIGHT-SOURCE, 2 FLUORESCENCE PHOTOMULTIPLIER TUBES, AND 2 LIGHT SCATTER DETECTORS

Multiple labeling is necessary for the detailed phenotyping of cells i n many biological systems in human and animal species. In a previous r eport, we described an approach permitting the study of three labels s imultaneously by using the two-color immunofluorescence and one light source (Mansour et al., Cytometry 11:636-641, 1990). This approach all owed enumeration of cell subpopulations positive for only one label an d negative for many others (X(+)A(-)B(-)...). We here present an impro vement of the previous approach to allow analysis of double positive p henotypes (X(+)Y(+)A(-)B(-)...), using only two fluorescence photomult iplier tubes and light scatter detectors, It consists of a two-step an alysis that does not require additional material than that used in the former technique. Briefly, all antibodies are conjugated to only two flurochromes: either FITC or PE. For the analysis of the X(+)Y(+)A(-)B (-)... phenoytpe, the Y, A and B labels are all coupled to the same dy e (FITC, e.g.) and the X label to the other dye (i.e, PE). In a first step, cells are labeled with X, A, and B, and in a second step, the se cond positive (Y) label is added. Two examples are supplied: CD56(+)CD 2(+)CD3(-)CD16(-) decidua infiltrating cells and CD3(+)TCR partial der ivative(+)CD4(-)CD8(-) peripheral blood lymphocytes. This technique is useful for qualitative as well as quantitative analysis, with cytomet ers that do not have the appropriate hardware to do true three-color i mmunofluorescence analysis. (C) 1994 Wiley-Liss, Inc.