Quantification of effector/target conjugation involving natural killer (NK) or lymphokine activated killer (LAK) cells by two-color flow cytometry.
ABSTRACT 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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ABSTRACT: To develop an affinity peptide that binds to gastric cancer used for the detection of early gastric cancer. A peptide screen was performed by biopanning the PhD-12 phage display library, clearing non-specific binders against tumor-adjacent normal appearing gastric mucosa and obtaining selective binding against freshly harvested gastric cancer tissues. Tumor-targeted binding of selected peptides was confirmed by bound phage counts, enzyme-linked immunosorbent assay, competitive inhibition, fluorescence microscopy and semi-quantitative analysis on immunohistochemistry using different types of cancer tissues. Approximately 92.8% of the non-specific phage clones were subtracted from the original phage library after two rounds of biopanning against normal- appearing gastric mucosa. After the third round of positive screening, the peptide sequence AADNAKTKSFPV (AAD) appeared in 25% (12/48) of the analyzed phages. For the control peptide, these values were 6.8 ± 2.3, 5.1 ± 1.7, 3.5 ± 2.1, 4.6 ± 1.9 and 1.1 ± 0.5, respectively. The values for AAD peptide were statistically significant (P < 0.01) for gastric cancer as compared with other histological classifications and control peptide. A novel peptide is discovered to have a specific binding activity to gastric cancer, and can be used to distinguish neoplastic from normal gastric mucosa, demonstrating the potential for early cancer detection on endoscopy.World Journal of Gastroenterology 05/2012; 18(17):2053-60. · 2.55 Impact Factor
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ABSTRACT: Hydroethidine (HE; or dihydroethidium) is the most popular fluorogenic probe used for detecting intracellular superoxide radical anion. The reaction between superoxide and HE generates a highly specific red fluorescent product, 2-hydroxyethidium (2-OH-E(+)). In biological systems, another red fluorescent product, ethidium, is also formed, usually at a much higher concentration than 2-OH-E(+). In this article, we review the methods to selectively detect the superoxide-specific product (2-OH-E(+)) and the factors affecting its levels in cellular and biological systems. The most important conclusion of this review is that it is nearly impossible to assess the intracellular levels of the superoxide-specific product, 2-OH-E(+), using confocal microscopy or other fluorescence-based microscopic assays and that it is essential to measure by HPLC the intracellular HE and other oxidation products of HE, in addition to 2-OH-E(+), to fully understand the origin of red fluorescence. The chemical reactivity of mitochondria-targeted hydroethidine (Mito-HE, MitoSOX red) with superoxide is similar to the reactivity of HE with superoxide, and therefore, all of the limitations attributed to the HE assay are applicable to Mito-HE (or MitoSOX) as well.Free radical biology & medicine 04/2010; 48(8):983-1001. · 5.42 Impact Factor
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ABSTRACT: The lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes. To test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro. The large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes. The large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support.Haematologica 02/2012; 97(7):994-1002. · 5.94 Impact Factor