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.
"The Fmoc group was removed in 20 % piperidine/DMF. FITC labeled peptide was synthesized by incubation of peptide (AcWHWTWLSEYK) with fluorescein-4-isothiocyanate (FITC) . Peptides were purified by reversed phase HPLC (JASCO, Tokyo, Japan) with a gradient of acetonitrile/0.1 % TFA; purity as estimated by HPLC was >95 %. "
[Show abstract][Hide abstract] ABSTRACT: A Gb3-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb3 antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb3 antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb3 antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb3 mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb3Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb3 mimic peptide potentially has great promise for use against Stxs.
"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 . Next, standard procedures to facilitate effector–target conjugation were performed [7–9,16]. "
[Show abstract][Hide abstract] ABSTRACT: In earlier work, we established a mathematical model to characterize the binding properties of cytotoxic cells to target cells. These properties can be described by the values of the maximum effector and target conjugate frequencies, alpha(max) and beta(max), respectively, and the dissociation constant of the conjugates formed, K(D) (Garcia-Peñarrubia, P., Cabrera, L., Alvarez, R., and Galvez, J., J. Immunol. Methods 155 (1992) 133). Here, we address the problem of exploring the physical meaning of these parameters and their relationships with cytotoxicity. With this purpose, conjugation between a human leukemic NK cell line (NKL) and K562 tumor cells has been studied from binding isotherms obtained from data of effector (alpha) and target (beta) conjugate frequencies measured by flow cytometry analysis at different effector-to-target ratios (R). The results have been compared to those obtained after target cells treatment with monoclonal antibodies recognizing adhesion molecules ICAM-1 (CD54) and LFA-3 (CD58) (which are able to block some of the receptors implicated in conjugation), as well as with cholera toxin (CTX) that can modify the state of affinity of some adhesion molecules such as LFA-1 (CD11a/CD18). The results show that: (1) blocking adhesion receptors CD54 and CD58 on the surface of target cells leads to a significant decrease of alpha(max) and beta(max), indicating that these parameters are related to the density of expression of receptors implicated in effector-target adhesion; (2) treatment of effector cells with CTX induced an increase of K(D), demonstrating that this parameter is associated with the effector-target affinity of the system; and (3) parallel experiments of conjugation and cytotoxicity showed that effector-target affinity and saturability influence the cytotoxic activity of the effector population.
[Show abstract][Hide abstract] 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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