Simultaneous three color and electronic cell volume analysis with a single UV excitation source

We have adapted an Ortho ICP-22 flow cytometer (Ortho Instruments, Westwood MA) for the simultaneous measurement of three independent fluorochromes and cell volume. This has been accomplished by the addition of a third photomultiplier tube and the development of a new electronic cell volume (ECV) flow cell. Cells are first analyzed as they pass through the 100 U ECV aperture and are then excited approximately 15 musec later by the 365 nm mercury are beam reflected by a 400 nm dicroic mirror. Independent blue, green and red signals can be associated by a delay circuit to the ECV signal from the same cell. We have developed this system as an aid in the analysis of tumor cell and macrophage heterogeneity and differentiation. The choice of stain combinations to be used is extremely flexible and permits the analysis of a wide range of enzyme activities in conjunction with DNA/RNA and phagocytic probes. Data presented indicates the value of this approach in identifying the presence of plasminogen activator-like activity in both tumor and inflammatory cells within a malignant effusion as well as the quantitative expression of a number of markers of macrophage differentiation. Although the described techniques have been developed on a mercury arc instrument, they can be used equally well with cell sorters.