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Merritt, Dennis

doi:10.1007/978-1-4613-8911-8_6

Cited by 2 publications

(5 citation statements)

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“…In each case the resting [Ca2+]1 of around 100 nm was elevated to a transient peak of around 1 ,UM. These responses of fluo-3loaded cells are similar to those previously reported for fura-2loaded neutrophils (Merritt, 1989;Merritt et al, 1989) and platelets (Pollock et al, 1986;Merritt & Hallam, 1988).…”

Section: Fmin9supporting

confidence: 88%

“…Calibration of the traces for neutrophils resulted in values of [Ca2+]i very similar to those obtained in the presence of buffer with either fluo-3 (Fig. 4a) or with fura-2 (Merritt, 1989;Merritt et al, 1989). It was not possible to use heparinized plasma for the experiments with platelets, because heparin inhibits platelet responses; citrated plasma therefore had to be used.…”

Section: Fmin9supporting

confidence: 69%

“…Since the introduction of fura-2, it has been used to measure [Ca2+Ji in many cell types (e.g. Tsien & Poenie, 1986), including platelets (Pollock et al, 1986;Merritt & Hallam, 1988;Sage et al, 1989) and neutrophils (Merritt, 1989;Merritt et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

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Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+

Merritt¹,

McCarthy²,

Davies³

et al. 1990

Biochemical Journal

284

108

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A description is given of the methodology, and problems encountered, for the use of a new fluorescent Ca2(+)-indicator dye, fluo-3, in neutrophils and platelets. The higher Kd and longer excitation wavelength of fluo-3 can have significant advantages over fura-2. Although neutrophils and platelets are used as examples, these observations will be applicable to other cell types. The Kd of fluo-3 for binding Ca2+ at 37 degrees C was measured and found to be 864 nM; the previously published value was 400 nM at 22 degrees C. The Kd of fluo-3, like that of fura-2, is therefore very temperature-dependent. Protocols for loading cells, and preventing leakage of fluo-3, are described; probenecid, known to inhibit fura-2 leakage from cells, was found to be essential to get good fluo-3 signals from platelets. Calibration of fluo-3 fluorescence signals to [Ca2+] and methods for obtaining maximum and minimum fluorescence signals are described; these methods differ from those used with fura-2. Agonist-stimulated responses of fluo-3-loaded neutrophils and platelets are shown, and the calculated cytosolic [Ca2+] is comparable with that previously obtained with fura-2. Responses of cells in the presence of plasma are also shown; such measurements, unobtainable with quin2, fura-2 or indo-1, are possible with fluo-3, owing to its longer excitation wavelengths. Co-loading of cells with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid and fluo-3 is included as an example of how cytosolic [Ca2+] can be buffered and manipulated. Many of these observations will be of value when using fluo-3 (or other Ca2(+)-indicator dyes) in most cell types.

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Section: Fmin9supporting

confidence: 88%

Section: Fmin9supporting

confidence: 69%

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Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+

Merritt¹,

McCarthy²,

Davies³

et al. 1990

Biochemical Journal

284

108

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“…Stimulation of human neutrophils with various agonists including chemotactic peptide (fMet-Leu-Phe), platelet-activating factor (PAF) and ATP results in an increase in the cytosolic Ca2l concentration [Ca2+]i as measured by the use of fluorescent Ca2" indicator dyes [1][2][3][4][5][6][7][8]. Agonist-evoked rises in [Ca2"], are due to both Ca2" release from intracellular stores and Ca2" entry from the extracellular medium.…”

Section: Introductionmentioning

confidence: 99%

“…Agonist-evoked rises in [Ca2"], are due to both Ca2" release from intracellular stores and Ca2" entry from the extracellular medium. The release of Ca2" from intracellular stores is indicated by the ability of agonists to elevate [Ca2+]i in the absence of extracellular Ca2" [1][2][3][4][5][6][7][8]. An additional influx component is apparent from the following studies.…”

Section: Introductionmentioning

confidence: 99%

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils

Sage

Pintado

Mahaut‐Smith

et al. 1990

Biochemical Journal

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The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.

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Cited by 2 publications

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Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+

Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils

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