SIMULTANEOUS MEASUREMENT OF 5-CELL SURFACE-ANTIGENS BY 5-COLOR IMMUNOFLUORESCENCE

Multicolour immunofluorescence and flow cytometry were used for simult aneous measurement of five-cell surface antigens on murine spleen cell s. We have been able to quantitate T-cells, T-cell subsets, B cells, a nd expression of the activation marker I-Ad from a single sample using four directly conjugated monoclonal antibodies LYT2-APC, L3T4-PE, B22 0-RED613, I-Ad-FITC and one indirect step THY1.2-biotin/streptavidin-C ascade Blue. Three excitation wavelengths were used (488 nm, 647 nm, a nd U.V. 351-364 nm) for fluorescence measurements. The combination of fluorochromes used provided good resolution such that all five fluores cence signals were spectrally resolved. The percentage of cells positi ve for expression of a specific cell surface marker were almost identi cal for single-colour samples and the five-colour analysis, differing by only 0.3-1.5 percentage points. (C) 1994 Wiley-Liss, Inc.