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

Department of Chemistry, Oakland University, Rochester, MI 48309.
Journal of Immunological Methods (Impact Factor: 1.82). 07/1991; 139(2):281-92. DOI: 10.1016/0022-1759(91)90199-P
Source: PubMed


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 visual estimation of the frequency of fresh or IL-2-activated human lymphocytes that form 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 (alpha 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.

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    Biochimica et Biophysica Acta 07/2006; 1760(6):883-9. DOI:10.1016/j.bbagen.2006.03.018 · 4.66 Impact Factor
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    • "Briefly, effector cells were labeled green with 400 nM calcein acetoxymethylester Ca-AM (Molecular Probes, Eugene, OR) and target cells were labeled red with 253 lM hydroethidine HE (Molecular Probes, Eugene, OR). Both red and green dyes can be excited at a wavelength of 488 nm from an argon laser and neither affects cell viability nor conjugate formation [15]. Next, standard procedures to facilitate effector–target conjugation were performed [7–9,16]. "
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    Cellular Immunology 03/2002; 215(2):141-50. DOI:10.1016/S0008-8749(02)00023-0 · 1.92 Impact Factor
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    ABSTRACT: The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector-target conjugates involving cloned human natural killer (NK) or interleukin-2 (IL-2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one-fourth of conjugates between K-562 cells and human PBL that had been cultured with or without IL-2 by this treatment. The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector-target conjugates are incubated at 37°C in the absence of calcium—thereby precluding lysis—the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.
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