Flow cytometric measurement of [Ca2+]i and pHi in conjugated natural killer cells and K562 target cells during the cytotoxic process

Graft, M. van; Kraan, Yvonne M.; Segers-Nolten, Ine; Radošević, Katarina; Grooth, B.G. de; Greve, Jan

doi:10.1002/cyto.990140304

Cited by 21 publications

(15 citation statements)

References 13 publications

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“…Flow cytometry has previously been used to measure calcium flux in other cell types, including eosinophils, lymphocytes, natural killer cells, and platelets [15][16][17][18]. Here we describe that calcium flux can be detected also in basophils contained in mixed PBMC preparations and double stained with CD123 and HLA-DR. Until now, it was impossible to measure calcium flux in native basophils, as these cells are rare in blood, and conventional methods such as fluorescence spectrometry rely on large amounts of purified cells.…”

Section: Discussionmentioning

confidence: 67%

A Novel Assay to Measure the Calcium Flux in Human Basophils: Effects of Chemokines and Nerve Growth Factor

Heinemann

Ofner²,

Amann³

et al. 2002

Pharmacology

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The calcium flux in human basophils was measured by flow cytometry. Peripheral blood mononuclear cells were labeled with anti-CD123 and anti-HLA-DR antibodies, loaded with fluo-3 acetoxymethyl ester (2 µmol/l) in the presence of probenecid (2.5 µmol/l) and Pluronic F-127 (0.02%) for 20 min, and equilibrated with Ca²⁺ (1.8 mmol/l) and Mg²⁺ (1 mmol/l) for 5 min. The levels of intracellular free calcium were monitored as changes in fluorescence. Cross-linking of surface IgE on basophils with anti-IgE antibodies caused effective calcium flux in atopic, but not in healthy, donors. Concentration-dependent responses to monocyte chemoattractant protein 1 (MCP-1), eotaxin, macrophage inflammatory protein 1 alpha (MIP-1α), and C5a (0.3–10 nmol/l) were observed in all subjects, with a rank order of potency of C5a = MCP-1 > eotaxin > MIP-1α. In contrast, the rank order of potency in causing basophil shape change (i.e., increase in forward scatter) was eotaxin > C5a > MCP-1 > MIP-1α. Nerve growth factor (NGF; 15 nmol/l) did not induce calcium flux in basophils, and pretreatment of cells with a low concentration of NGF (0.3 nmol/l), which has previously been shown to prime basophils for mediator release, had no effect on the calcium response to subsequent stimulation with C5a. We conclude that calcium mobilization is differentially involved in signaling to chemoattractants in basophils and that it is correlated with the agonist’s efficacy to induce mediator release. The data also suggest that priming of basophil responses by NGF does not rely on enhanced calcium mobilization.

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Section: Discussionmentioning

confidence: 67%

A Novel Assay to Measure the Calcium Flux in Human Basophils: Effects of Chemokines and Nerve Growth Factor

Heinemann

Ofner²,

Amann³

et al. 2002

Pharmacology

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“…Transmembrane stimulation of lymphocytes, at the G 0 -G 1 resting phase, is induced by specific antigens, mitogens, or antibodies to certain cell-surface molecules. The stimulation results in a complex series of well-characterized molecular events that culminate in lymphocyte activation, transformation, mitosis, and finally apoptosis (7)(8)(9). These events are associated with early changes in membrane potential coupled with an influx of Na ϩ and changes in pH, followed by the influx and internal release of Ca 2ϩ ions.…”

mentioning

confidence: 99%

Determination of individual cell Michaelis‐Menten constants

et al. 2001

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Background: A novel methodology for the measurement and analysis of apparent K M (Michaelis-Menten constant) and V MAX values of individual cells is suggested. It is based on a mathematical model that considers substrate influx into the cell, its intracellular enzymatic hydrolysis, and the product efflux. The mathematical formulation was approximated linearly in order to analyze intracellular substrate conversion characteristics via Michaelis-Menten theory.

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“…A change in the intracellular calcium homeostasis appears to be not only a hallmark of cell activation but also generally accompanies cell death (17). It has been observed that the intracellular calcium concentration ([ca' + la) increases in the cells that are attacked by the cytotoxic cells (1,19, 28). In some cases this increase appeared to be a prereq uisite for efficient cell killing (13,26).…”

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confidence: 99%

Changes in intracellular calcium concentration and pH of target cells during the cytotoxic process: A quantitative study at the single cell level

1995

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This study reports on the changes in intracellular calcium concentration ([Ca"],) and intracellular [pH], remains changed during the time the cells were followed (10 min). The programming time (i.e., the time from the initiation of the cytotoxic process to the time that a change in the physiological parameter was detected) of the killing process that leads to an immediate target cell death appears to be shortest at [pHla 7.3-7.6 (approximately 3 min). o 199s wuey-~lss, hc.

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Flow cytometric measurement of [Ca²⁺]_i and pH_i in conjugated natural killer cells and K562 target cells during the cytotoxic process

Cited by 21 publications

References 13 publications

A Novel Assay to Measure the Calcium Flux in Human Basophils: Effects of Chemokines and Nerve Growth Factor

A Novel Assay to Measure the Calcium Flux in Human Basophils: Effects of Chemokines and Nerve Growth Factor

Determination of individual cell Michaelis‐Menten constants

Changes in intracellular calcium concentration and pH of target cells during the cytotoxic process: A quantitative study at the single cell level

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