title: Multiple labeling using two-color immunofluorescence with only one light source, two fluorescence photomultiplier tubes, and two light scatter detectors.
abstract: 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.
authors.affiliation: Institut National de Transfusion Sanguine, Paris, France.
authors.forename: I
authors.initials: I
authors.lastname: Mansour
authors.forename: M A
authors.initials: MA
authors.lastname: Jarraya
authors.forename: P
authors.initials: P
authors.lastname: Gane
authors.forename: M F
authors.initials: MF
authors.lastname: Reznikoff
mesh_terms: D015703:Antigens, CD / Q000032:analysis*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D018801:CD2 Antigens; D017252:CD3 Complex / Q000032:analysis; D015704:CD4 Antigens / Q000032:analysis; D019002:CD56 Antigen; D016827:CD8 Antigens / Q000032:analysis; D003656:Decidua / Q000166:cytology / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008214:Lymphocytes / Q000166:cytology* / Q000276:immunology; D011247:Pregnancy; D017452:Receptors, IgG / Q000032:analysis; D011971:Receptors, Immunologic / Q000032:analysis
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D018801:CD2 Antigens; D017252:CD3 Complex; D015704:CD4 Antigens; D019002:CD56 Antigen; D016827:CD8 Antigens; D017452:Receptors, IgG; D011971:Receptors, Immunologic