Quantitation and sorting of vitally stained natural killer cell-target cell conjugates by dual beam flow cytometry

We have detected formation of stable associations, or conjugates, between fluorescein diacetate-(FDA) stained human natural killer (NK) cells and Hoechst 33342-(HO342) stained tumor cells by dual laser flow cytometry. Conjugates in mixtures of effectors and targets emitted both green (FDA) and blue (HO342) fluorescence. This was confirmed by cell sorting. More than 90% of the conjugates included one target and one effector cell. Conjugate formation frequency was temperature independent between 4 and 37 degrees C, optimized by 10 min, and stable for 1 hr. Enrichment of effector populations for cells mediating lysis of standard NK targets and for cells reacting with OKM1, Leu-7, and Leu-11b monoclonal antibodies also enriched conjugate-forming cells. Lysis of either OKM1+, Leu-11b+ effector subpopulations with antibody and complement eliminated, but treatment with these antibodies alone had no effect on, conjugate formation. Effector pretreatment with Leu-4 or 3A1 and complement increased the frequency of conjugation slightly. Flow-determined frequencies of NK-conjugate formation with 14 target cell lines correlated well with data derived from standard microscopic assays. However, the flow method was more rapid, could be used when target and effector were of comparable size, and permitted isolation of conjugates by sorting.