Measurement of DNA and antigen expression of live cells using three fluorochromes and two detectors

In a multiparameter flow cytometric study, the fluorescence from the two different dyes fluorescein and dansyl-lysine were detected in the same spectral interval on the same photomultiplier tube, using two lasers to excite the fluorochromes at two separate laser foci. The two signals were split by a T-cable at the output of the photomultiplier tube and synchronized by delaying the first signal. Dansyl-lysine binds to the membrane of heat-inactivated cells and was used to distinguish between live and dead cells after a hyperthermic treatment. By gating on the dansyl-lysine signal, the DNA distribution and surface antigen expression of live and dead cells were obtained separately, using Hoechst 33342 and a monoclonal antibody conjugated to fluorescein.