Multiple labeling using two-color immunofluorescence with only one light source, two fluorescence photomultiplier...

Multiple labeling is necessary for the detailed phenotyping of cells in many biological systems in human and animal species. In a previous report, we described an approach permitting the study of three labels simultaneously by using the two-color immunofluorescence and one light source (Mansour et al., Cytometry 11:636-641, 1990). This approach allowed enumeration of cell subpopulations positive for only one label and negative for many others (X+ A- B- ...). We here present an improvement of the previous approach to allow analysis of double positive phenotypes (X+ Y+ A- B- ...), using only two fluorescence photomultiplier tubes and light scatter detectors. It consists of a two-step analysis that does not require additional material than that used in the former technique. Briefly, all antibodies are conjugated to only two fluorochromes: either FITC or PE. For the analysis of the X+ Y+ A- B- ... phenotype, the Y, A and B labels are all coupled to the same dye (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 second positive (Y) label is added. Two examples are supplied: CD56+ CD2+ CD3- CD16- decidua infiltrating cells and CD3+ TCR delta+ CD4- CD8- peripheral blood lymphocytes. This technique is useful for qualitative as well as quantitative analysis, with cytometers that do not have the appropriate hardware to do true three-color immunofluorescence analysis.