Quantification of effector/target conjugation involving natural killer (NK) or lymphokine activated killer (LAK) cells by two-color flow cytometry

Precise estimates of the frequency of NK- and LAK-target conjugates were obtained by two-color flow cytometry using hydroethidine and calcein as intracellular labels for target cells and effector cells, respectively. These two dyes can easily be used with a standard single-laser flow cytometer with excellent signal separation and dye retention. Hydroethidine labeling did not alter target susceptibility, and calcein labeling did not significantly alter NK function. Excellent agreement was obtained between this flow cytometric method and cisual estimation of the frequency of fresh or IL-2-activated human lymphocytes that from conjugates with K-562 target cells. The percentage of cloned NK or LAK cells that form conjugates with K-562 target cells was dependent on the E : T ratio, with extrapolated maximum conjugate frequencies ( α max) of 40–50%. However, the frequency of lymphocytes forming conjugates with K-562 cells did not closely correlate with the cytolytic activity of a given lymphocyte population. This two color flow cytometric method employing a pair of fluorochromes that do not modify cell membranes or alter cell function in cytotoxicity assays should facilitate further studies of mechanisms involved in the initial stages of target cell recognition by NK and LAK cells.