Cell membrane labeling with fluorescent dyes for the demonstration of cytokine‐induced fusion between monocytes and tumor cells

Spötl, Ludwig; Sarti, Alessandra; Dierich, Manfred P.; Möst, Johannes

doi:10.1002/cyto.990210208

Cited by 40 publications

(28 citation statements)

References 21 publications

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“…Integration of the PKH2-GL-labeled liposomes with bacterial membranes was confirmed by flow cytometry as well. This association indicates a direct incorporation of liposomal phospholipids into the bacterial membranes because (i) the probe inserts its aliphatic carbon tails into membranes (15,38) and (ii) dissociation of the probe from the liposomes to the bacterial membrane via an aqueous environment is unlikely due to the strong hydrophobic nature of this probe. Further, we applied a centrifugation step with a sucrose cushion as well as successive PBS washes to rule out the possibility of liposomal adsorption to bacteria.…”

Section: Discussionmentioning

confidence: 98%

“…Several hypotheses including reduced electrostatic repulsion of liposomal antibiotics or protection of the drugs from bacterial enzymes may explain the mechanism of enhanced antimicrobial activities of liposomal formulations (36)(37)(38). We hypothesized that the enhanced antimicrobial activity of these formulations is due to their fusion with the bacterial outer membrane.…”

Section: Discussionmentioning

confidence: 99%

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Mechanism of Enhanced Activity of Liposome-Entrapped Aminoglycosides against Resistant Strains of Pseudomonas aeruginosa

Mugabe

Halwani

Azghani

et al. 2006

Antimicrob Agents Chemother

156

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Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of both free and liposomal drugs were evaluated. To define the mechanism of liposome-bacterium interactions, transmission electron microscopy (TEM), flow cytometry, lipid mixing assay, and immunocytochemistry were employed. Encapsulation of aminoglycosides into liposomes significantly increased their antibacterial activity against the resistant strains used in this study (MICs of >32 versus <8 g/ml). TEM observations showed that liposomes interact intimately with the outer membrane of P. aeruginosa, leading to the membrane deformation. The flow cytometry and lipid mixing assays confirmed liposome-bacterial membrane fusion, which increased as a function of incubation time. The maximum fusion rate was 54.3% ؎ 1.5% for an antibiotic-sensitive strain of P. aeruginosa and 57.8% ؎ 1.9% for a drug-resistant strain. The fusion between liposomes and P. aeruginosa significantly enhanced the antibiotics' penetration into the bacterial cells (3.2 ؎ 2.3 versus 24.2 ؎ 6.2 gold particles/bacterium, P < 0.001). Our data suggest that liposome-entrapped antibiotics could successfully resolve infections caused by antibiotic-resistant P. aeruginosa through an enhanced mechanism of drug entry into the bacterial cells.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Mechanism of Enhanced Activity of Liposome-Entrapped Aminoglycosides against Resistant Strains of Pseudomonas aeruginosa

Mugabe

Halwani

Azghani

et al. 2006

Antimicrob Agents Chemother

156

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“…An important caveat of membrane dyes is that if an unlabeled phagocytic cell ingests a labeled cell, a process involving membrane fusion, membrane dye can be transferred to phagocyte membranes [34]. The frequency with which this occurs in vivo has not been established, and many of the studies using membrane dyes as a tracking marker have not adequately controlled for this possibility.…”

Section: Membrane Dyesmentioning

confidence: 99%

Optimizing Techniques for Tracking Transplanted Stem Cells In Vivo

2005

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The potential for bone marrow-derived cells (BMDCs) to contribute to nonhematopoietic tissues has generated considerable debate in recent years. Causes for the controversies include disparities in the techniques used to track engraftment of BMDCs, inappropriate tissue preparation, a lack of appropriate positive and negative controls, and basic misunderstandings about how to properly collect and interpret images from epifluorescent and confocal microscopes. Our laboratory was among the first to use bone marrow transplants from transgenic mice constitutively expressing enhanced green fluorescent protein (GFP) to study the ability of BMDCs to give rise to nonhematopoietic tissue types, a system that is now in widespread use. During our 6 years of experience using GFP, as well as beta-galactosidase and the Y chromosome, to track BMDCs in vivo, we have identified many difficulties and have developed techniques to resolve them. We discuss several of these methods, and, in particular, we describe ratiometric analysis techniques for improving detection of transplanted cells derived from genetically modified bone marrow. Finally, to help resolve reported discrepancies regarding the frequency with which BMDCs contribute to skeletal myofibers, we demonstrate that the pattern of highly autofluorescent myofibers in skeletal muscle is clearly distinct from that of GFP-expressing myofibers and describe how unambiguous conclusions can be drawn from such data. Stem Cells

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“…In the present study we labeled sporozoites of Eimeria tenella with a new uorescent cell linker dye, wherein a stable reporter molecule is incorporated into the membrane of target cells allowing them to be tracked in vitro or in vivo. Cell linker dyes have been used previously in cytotoxicity assays (Embleton et al 1991;Kroesen et al 1992), and to study membrane dynamics in cell fusion studies (Spotl et al 1995). We found that PKH-67 GL used within the guidelines recommended by the manufacturer produces a bright green¯uorescent labeling of Eimeria sporozoites, facilitating observation of intracellular parasites and analysis by¯ow cytometry.…”

Section: Discussionmentioning

confidence: 99%

Cell membrane labeling of Eimeria tenella sporozoites with the fluorescent dye PKH-67 GL for tracking parasite-host interactions

Lr²

2001

Parasitology Research

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The fluorescent cell linker dye PKH-67 GL was used as a vital stain for sporozoites of Eimeria tenella for tests on viability, invasion of cultured primary chick kidney cells, flow cytometric analysis and fluorescence microscopy. The effect of PKH-67 GL on sporozoites was tested at a range of concentrations of dye and sporozoites. In flow cytometric analysis, 0.5-40x10(-6) M of PKH-67 GL labeled sporozoites to some degree, with the percentage of labeled sporozoites increasing with higher dye concentrations. The optimum concentration was 2x10(-6) M, allowing easy observation by fluorescence microscopy. Morphological changes in the sporozoite at concentrations greater than 5x10(-6) M were accompanied by loss of viability according to a propidium iodide inclusion assay. Sporozoite penetration of primary chick kidney cells was unaffected by the optimal level of 2x10(-6) M, allowing observation of intracellular activities. Overall, the cell linker dye greatly facilitated observation of E. tenella in vitro and in flow cytometric analysis.

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Cell membrane labeling with fluorescent dyes for the demonstration of cytokine‐induced fusion between monocytes and tumor cells

Cited by 40 publications

References 21 publications

Mechanism of Enhanced Activity of Liposome-Entrapped Aminoglycosides against Resistant Strains of Pseudomonas aeruginosa

Mechanism of Enhanced Activity of Liposome-Entrapped Aminoglycosides against Resistant Strains of Pseudomonas aeruginosa

Optimizing Techniques for Tracking Transplanted Stem Cells In Vivo

Cell membrane labeling of Eimeria tenella sporozoites with the fluorescent dye PKH-67 GL for tracking parasite-host interactions

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