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, LM‐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 singlecolour samples and the five‐colour analysis, differing by only 0.3–1.5 percentage points. © 1994 Wiley‐Liss, Inc.