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  • 1
    Electronic Resource
    Electronic Resource
    Fluorescence analysis of the size of a binding pocket of a peptide receptor at natural abundance (1990)
    s.l. : American Chemical Society
    Biochemistry 29 (1990), S. 313-316 
    Details
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1021/bi00454a002
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  • 2
    Electronic Resource
    Electronic Resource
    Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye (1980)
    Springer
    The journal of membrane biology 52 (1980), S. 257-272 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Changes in the fluorescence intensity of the dye 3-3′ dipentyloxacarbocyanine were measured in suspensions of purified human peripheral blood polymorphonuclear leukocytes (PMNs) during exposure to the chemotactic factors N-formyl-methionylleucyl-phenylalanine (f-met-leu-phe) and partially purified C5a. Incubation of PMNs with dye resulted in a stable fluorescence reflecting the resting membrane potential of the cell. Exposure of PMNs to dye did not affect stimulated chemotaxis or secretion. The mechanism of cell-associated dye fluorescence involved solvent effects from partitioning of the dye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5×10−9 m or greater) produced a biphasic response characterized as a decrease followed by an increase in fluorescence. No fluorescence response was seen in lysed PMNs, and no response was elicited by an inhibitor of f-met-leu-phe binding (carbobenzoxy-phenylalanyl-methionine). The ability of several other synthetic peptides to elicit a fluorescence response corresponded to their effectiveness as chemotactic agents. Although the first component of the response suggested a depolarization, it was not influenced by variation in the external concentration of sodium, potassium, chloride, or calcium, and could not be characterized as a membrane potential change. The second component of the response, which was inhibited by both Mg2+ (10mm)-EGTA (10mm) and high external potassium, was compatible with a membrane hyperpolarization. The data indicate that chemotactic factors produce changes in dye fluorescence which can, at least in part, be attributed to a hyperpolarizing membrane potential change occurring across the plasma membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01869194
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  • 3
    Electronic Resource
    Electronic Resource
    Separation and function of neutrophil karyogranuloplasts and comparison with cytoplasts and intact cells (1987)
    Springer
    Inflammation 11 (1987), S. 289-307 
    Details
    ISSN: 1573-2576
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Since neutrophjl cytoplasts lacking nucleus and granules were first prepared by centrifuging neutrophils over a discontinuous Ficoll gradient containing cytochalasin B, several functional deficits have been reported in these cytoplasts. Although these functional deficits have been considered to originate from the absence of organelles, cell damage during preparation could not be excluded. Therefore, in the following experiments the Ficoll gradient was modified to isolate both cytoplasts and karyogranuloplasts, which have a nucleus and granules and represent the cell after loss of the cytoplast. Electron microscopy and analysis of marker proteins and cell volume showed that karyogranuloplasts were distinct from neutrophils. The phorbol myristate acetate (PMA) orN-formylmethionylleucylphenylalanine (FMLP) -induced O 2 − release, corrected by surface area, was in the following order: neutrophils 〉 cytoplasts 〉 karyogranuloplasts. Both aggregation and membrane potential depolarization were maximal in neutrophils, intermediate in karyogranuloplasts, and lowest in cytoplasts when either PMA or FMLP was used as a stimulant. Extracellular release of the granule contents (degranulation) was triggered by FMLP in both neutrophils and karyogranuloplasts. Cytochalasin B pretreatment greatly enhanced FMLP-induced O 2 − release, degranulation, aggregation, and depolarization of membrane potential in neutrophils and karyogranuloplasts, but not in cytoplasts. The ability of cytochalasin B to potentiate FMLP-triggered cell function probably depends on granules or cell organelles which are depleted in cytoplasts. Chemokinesis and chemotaxis were impaired in both karyogranuloplasts and cytoplasts. Specific FML[3H]P binding was greater in karyogranuloplasts than in cytoplasts. Cellular actin content, measured by the DNase I inhibition assay, was abundant in cytoplasts and was extremely low in karyogranuloplasts. Karyogranuloplasts retain various neutrophil functions, except for chemotaxis, and provide an important control when studying the role of cell organelles in cytoplast function.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00915834
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  • 4
    Electronic Resource
    Electronic Resource
    Comparison of indirect probes of membrane potential utilized in studies of human neutrophils (1983)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 115 (1983), S. 105-115 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Four indirect probes of membrane potential, triphenylmethylphosphonium ion (TPMP+), 3,3′dipentyloxacarbocyanine [di-O-C5(3)], 3,3′ dipentylindocarbocyanine [di-I-C5(3)], and 3,3′ dipropylthiodicarbocyanine [di-S-C3(5)] have been used to study neutrophil (PMN) activation. The data extend previous studies indicating that the cyanine dye di-S-C3(5) not only exhibits a different fluorescence response mechanism from di-O-C5(3) [and di-I-C5(3)] but also that the fluorescence of di-S-C3(5) is destroyed by reactive oxygen products produced by neutrophils following stimulation. When these aspects of the probes are taken into account, the interpretations of the results using all three cyanine dyes are identical. Studies with the isotope TPMP+ indicate that long incubations are necessary for PMN to fully equilibrate during which time the PMNs depolarize. Use of TPB-, to shorten the TPMP+ equilibration time, produces results identical with those obtained using the cyanine dyes. The cyanine dyes and TPMP+/TPB- are toxic to neutrophil functions although they do not cause cell death. Toxicity can be avoided by using low concentrations of di-O-C5(3) and di-I-C5(3) but cannot be avoided with di-S-C3(5) or TPMP+/TPB-. Using di-O-C5(3) with the fluorescence-activated cell sorter, we demonstrate that heterogeneity of neutrophil responsiveness confuses the interpretation of studies characterizing the ionic basis of the fluorescence responses stimulated by certain stimuli. We conclude that some of the discrepancies currently reported in the literature using these probes are not due to inherent differences in the ability of the different probes to monitor the same event (i.e., PMN membrane potential) but instead are due to failure to correct for probe-specific problems or response heterogeneity.
    Additional Material: 11 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041150202
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  • 5
    Electronic Resource
    Electronic Resource
    Voltage-sensitive cyanine dye fluorescence signals in lymphocytes: Plasma membrane and mitochondrial components (1985)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 125 (1985), S. 61-71 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The origin of the cyanine dye fluorescence signal in murine and human peripheral blood leukocytes was investigated using the oxa- and indo-carbo-cyanines di-O-C5(3). and di-l-C5(3). Fluorescence signals from individual cells suspended with nanomolar concentrations of the dyes were measured in a flow cytometer modified to permit simultaneous four-parameter analysis (including two-color fluorescence or fluorescence polarization measurements). The contributions of mitochondrial membrane potential (Ψm) and plasma membrane potential (Ψpm) to the total voltage-sensitive fluorescence signal were found to depend on the equilibrium extracellular dye concentration, manipulated in these experiments by varying the ratio of dye to cell density. Hence, conditions could be chosen that amplified either the Ψm or the Ψpm component. Selective depolarization of lymphocytes or polymorphonuclear leukocytes (PMN) in mixed cell suspensions demonstrated that defining the partition of dye between cells and medium is requisite to assessing the heterogeneity of cell responses by cyanine dye fluorescence. At extracellular dye concentrations exceeding 5 nM in equilibrated cell suspensions, both mitochondrial and plasma membrane dye toxicity were observed. In murine splenic lymphocytes, plasma membrane toxicity (dye-induced depolarization) was selective for the B lymphocytes. Certain problems in calibration of Ψpm with valinomycin at low dye concentrations and perturbations of Ψpm by mitochondrial inhibitors are presented. These findings address the current controversy concerning Ψm and Ψpm measurement in intact cells by cyanine dye fluorescence. The finding of selective toxicity at low cyanine dye concentrations suggest that purported differences in resting Ψm among cells or changes in Ψpm with cell activation may reflect varible susceptibility to dye toxicity rather than intrinsic cell properties.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041250109
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