Simultaneous measurement of five-cell surface antigens by five-colour immunofluorescence.
Multicolour immunofluorescence and flow cytometry were used for simultaneous measurement of five-cell surface antigens on murine spleen cells. We have been able to quantitate T-cells, T-cell subsets, B cells, and expression of the activation marker I-Ad from a single sample using four directly conjugated monoclonal antibodies LYT2-APC, L3T4-PE, B220-RED613, I-Ad-FITC and one indirect step THY1.2-biotin/streptavidin-Cascade Blue. Three excitation wavelengths were used (488 nm, 647 nm, and U.V. 351-364 nm) for fluorescence measurements. The combination of fluorochromes used provided good resolution such that all five fluorescence signals were spectrally resolved. The percentage of cells positive for expression of a specific cell surface marker were almost identical for single-colour samples and the five-colour analysis, differing by only 0.3-1.5 percentage points.
Beavis
A J
AJ
Department of Immunology, Schering Plough Research Institute, Kenilworth, New Jersey 07033.
Pennline
K J
KJ
D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis*; D000950:Antigens, Ly / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D000949:Histocompatibility Antigens Class II / Q000032:analysis*; D016130:Immunophenotyping / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000032:analysis; D008213:Lymphocyte Activation; D016131:Lymphocyte Subsets; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D013154:Spleen / Q000166:cytology; D018800:Thy-1 Antigens
D000944:Antigens, Differentiation, B-Lymphocyte; D000945:Antigens, Differentiation, T-Lymphocyte; D000950:Antigens, Ly; D000954:Antigens, Surface; D005456:Fluorescent Dyes; D000949:Histocompatibility Antigens Class II; D008562:Membrane Glycoproteins; D018800:Thy-1 Antigens; D017493:Leukocyte Common Antigens