Rare-event sorting by fixed-time flow cytometry based on changes in intracellular free calcium

Tárnok, Attila

doi:10.1002/(sici)1097-0320(19970101)27:1<65::aid-cyto8>3.0.co;2-k

Cited by 8 publications

(3 citation statements)

References 16 publications

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“…In order to largely exclude dead cells and debris from measurement, an appropriate FSC/SSC gate was already applied during data acquisition. Under certain conditions, oxonol attachment to the tube system of the flow cytometer has to be considered (51). Measurement of cells stained with 25 nM DiBAC 4 (3) preceded by rinsing with 25,000 nM (the highest concentration used for cell staining in this study) led to a threefold enhancement in fluorescence for the first and still 2.4‐fold for the second sample due to dye release from the tube system of the flow cytometer.…”

Section: Methodsmentioning

confidence: 99%

Methodological aspects of measuring absolute values of membrane potential in human cells by flow cytometry

et al. 2009

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The bis-barbituric acid oxonol, DiBAC 4 (3) is used as a standard potentiometric probe in human cells. However, its fluorescence depends not only on membrane potential but also varies with nonpotential related changes in the amount of intracellular free and bound dye. This study demonstrates the influence of different experimental conditions on this nonspecific fluorescence proportion. IGR1 melanoma cells as a model were specifically altered in cell volume and protein content by depolarizing treatments or cell cycle synchronization. Flow cytometry was performed over a wide range of extracellular DiBAC 4 (3) concentrations. Fixation and increase in protein content led to a nonspecifically enhanced fluorescence, while changes in the amount of free intracellular dye as a result of altered cell volume proved to be negligible. To establish a calibration curve using totally depolarized cells, the pore-forming action of gramicidin should be preferred to fixation. Below 100 nM DiBAC 4 (3), the logarithmic relation between cell fluorescence and dye concentration turned into a virtually linear function intersecting with zero. Consequently, calibration can then be confined to determination of the fluorescence of depolarized cells stained with the same concentration as used for the actual measurement of membrane potential. Unexpectedly, quenching of fluorescence occurred in totally depolarized cells at concentrations higher than 6,250 nM. Linearity and quenching could be confirmed by additional experiments on Chinese hamster ovary CHO-K1 and B lymphoblastoid LCL-HO cells. ' International Society for Advancement of CytometryKey terms bis-barbituric acid oxonol; flow cytometry; membrane potential; mV-scale calibration PATCH-clamp recording is considered to be the reference method to quantitatively measure the plasma membrane potential V p in human cells. A disadvantage in respect of routine use is that this method is time-consuming and laborious thus usually resulting in measuring values for only a small number of cells (1,2). Therefore, voltage-sensitive anionic and cationic dyes, which partition between the extracellular staining buffer and the cytoplasm under the influence of V p according to a Nernstian distribution, have been applied as an alternative (3-12).Due to their strong accumulation in mitochondria (13,14), cationic dyes report V p alone only when the mitochondrial membrane potential V m remains constant during the measurement. To ensure this condition, V m was specifically abolished by ionophores in some studies (15)(16)(17). With increasing concentration, the monomer form of cationic dyes, among them carbocyanines as the most popular dye class, is usually more and more displaced by dimers or higher aggregates. This process takes place both with dye dissolved in aqueous solution or bound to cell compartments, and is accompanied by distinct spectral shifts in absorbance and fluorescence maxima leading to quenching of the fluorescence of the monomers (18-21). For example, DiSC 3 (5) monomers show red flu...

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Section: Methodsmentioning

confidence: 99%

Methodological aspects of measuring absolute values of membrane potential in human cells by flow cytometry

et al. 2009

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“…A range of methodologies have been developed for analysis of changes in intracellular calcium concentration in cell populations using cell-permeable calcium-sensitive fluorescent dyes (6)(7)(8)(9)(10)(11). Most flow cytometers have a pressurized system for aspirating the sample in which the tubes must be sealed, and therefore, there is no opportunity for continuous addition of test compounds to the cell suspension.…”

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confidence: 99%

A flow‐cytometric method for continuous measurement of intracellular Ca²⁺ concentration

2010

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Alterations in intracellular Ca 21 concentration are amongst the most rapid responses to a variety of stimuli in mammalian cells. In the nervous system in particular, responses occur within nanoseconds. A major challenge in intracellular Ca 21 analysis is to achieve measurements within this very fast time frame. To date, the dynamic intracellular Ca 21 concentration has been monitored by confocal microscopy, plate-based assays, spectrofluorometry, and flow cytometry, although there are issues with the number of cells analyzed or gaps in recording due to the addition of compounds, with significant loss of detail of a rapid Ca 21 response. The new generation of flow cytometers (such as Accuri C6) resolves this problem by allowing the addition of test compounds with continuous monitoring of thousands of cells, providing a method for dynamic Ca 21 measurements. This system was tested with commonly used Ca 21 modulating agents in C6 glioma cells. Thapsigargin (TG), a blocker of Ca 21 uptake into the endoplasmic reticulum (ER), causes a significant increase in the intracellular calcium concentration via ER emptying followed by Ca 21 entry via store-operated Ca 21 channels (SOCC). This well-established pathway can be partially inhibited by 2-aminoethoxydiphenyl borate (2-APB), a blocker of SOCC. Both the increase with TG alone and the partial increase when coincubated with 2-APB were observed with continuous recording along with calibration curves using an Accuri C6 flow cytometer. With these new cytometers, dynamic Ca 21 concentration measurement becomes extremely accessible and accurate, while also providing extensive and valuable data regarding population health and responsiveness. ' AN alteration in intracellular calcium concentration is one of the most common second messenger responses now known in mammalian cells. These responses have been shown to play a critical role in long established mechanisms such as action potential generation in electrically excitable cells (1,2) and have been implicated in a wide variety of intracellular signaling and regulatory pathways, including initiation of apoptosis (3), alteration of cell surface protein expression (4), and response to mechanical stress on the plasma membrane (5). The vast majority of changes in intracellular calcium is extremely rapid and occurs within a nanosecond timescale. Therefore, if the full implications of a change in the intracellular calcium concentration are to be understood, then a protocol for extremely rapid and sensitive intracellular calcium determination is required.Methods currently in use for intracellular calcium determination include confocal microscopy, plate-based assays, spectrofluorometry, and flow cytometry. Although confocal microscopy examines in great detail the intracellular calcium dynamics over time, the number of cells examined per field is low, and therefore, much of the response throughout a population of cells may be unrecorded. Plate-based assays overcome this issue by recording the response of the entire population...

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“…Besides being regulated during the ontogenesis of cardiovascular and urinary systems, a large set of evidence exists showing that modulation of B2 receptor expression and function also appears during neuronal development. Thus, it has been detected in central and peripheral noradrenergic neurons, in the spinal cord, in neuronal differentiating PC12 pheochromocytoma cells, and in neuroblastoma and glia-derived cell lines (12)(13)(14)(15)(16)(17)(18). A cross-talk between the B2BKR and other hormone and neurotransmitter receptors involved in neuronal development and function has been reported, such as endothelin, serotonin, and purinergic receptors (19,20).…”

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Neuronal Differentiation of P19 Embryonal Carcinoma Cells Modulates Kinin B2 Receptor Gene Expression and Function

Martins

Resende

Majumder

et al. 2005

Journal of Biological Chemistry

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Kinins are vasoactive oligopeptides generated upon proteolytic cleavage of low and high molecular weight kininogens by kallikreins. These peptides have a well established signaling role in inflammation and homeostasis. Nevertheless, emerging evidence suggests that bradykinin and other kinins are stored in the central nervous system and may act as neuromediators in the control of nociceptive response. Here we show that the kinin-B2 receptor (B2BKR) is differentially expressed during in vitro neuronal differentiation of P19 cells. Bradykinin (BK)1 and kallidin are biological active peptides generated by the proteolytic cleavage of kininogens by serine proteases of the kallikrein family. High molecular weight kininogens are precursors of BK, whereas low molecular weight kininogens give origin to kallidin. Along with other kinins, BK and kallidin elicit a wide range of physiological responses, being classically involved in the control of cardiovascular homeostasis and inflammation. As a matter of fact, altered function of the kallikrein-kinin system has been implicated in the development of various pathological conditions such as arthritis, pancreatitis, and asthma (for a review see Refs. 1 and 2). Emerging evidence shows that kinins are stored in neuronal cells of the central nervous system and may act as neuromediators in various functions, including the control of nociceptive information (for a review see Ref.3). Expression of kallikrein in developing rat brains (4) supports the notion that kinin-induced receptor activity might be required during neuronal development. BK has also been shown to enhance the release of neurotransmitters such as noradrenalin and neuropeptide Y by sympathetic neurons, chromaffin cells, and pheochromocytoma cells (5-8). Moreover, BK implication in the control of calcium homeostasis has already been demonstrated in adult sensory neurons (9).Most of the biological actions of BK and kallidin are mediated by a serpentine receptor coupled to a G-protein, the kinin-B2 receptor (B2BKR), which is constitutively expressed and widely distributed throughout central and peripheral tissues under physiological conditions. However, there is evidence that expression of B2BKRs during development is regulated. For instance, B2BKR expression has been shown to be involved in the development of the urinary and cardiovascular systems (10). Inhibition of B2BKR activity in rat embryos resulted in animals with disturbed kidney development (11). Besides being regulated during the ontogenesis of cardiovascular and urinary systems, a large set of evidence exists showing that modulation of B2 receptor expression and function also appears during neuronal development. Thus, it has been detected in central and peripheral noradrenergic neurons, in the spinal cord, in neuronal differentiating PC12 pheochromocytoma cells, and in neuroblastoma and glia-derived cell lines (12-18). A cross-talk between the B2BKR and other hormone and neurotransmitter

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Rare-event sorting by fixed-time flow cytometry based on changes in intracellular free calcium

Cited by 8 publications

References 16 publications

Methodological aspects of measuring absolute values of membrane potential in human cells by flow cytometry

Methodological aspects of measuring absolute values of membrane potential in human cells by flow cytometry

A flow‐cytometric method for continuous measurement of intracellular Ca²⁺ concentration

Neuronal Differentiation of P19 Embryonal Carcinoma Cells Modulates Kinin B2 Receptor Gene Expression and Function

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Rare-event sorting by fixed-time flow cytometry based on changes in intracellular free calcium

Cited by 8 publications

References 16 publications

Methodological aspects of measuring absolute values of membrane potential in human cells by flow cytometry

Methodological aspects of measuring absolute values of membrane potential in human cells by flow cytometry

A flow‐cytometric method for continuous measurement of intracellular Ca2+ concentration

Neuronal Differentiation of P19 Embryonal Carcinoma Cells Modulates Kinin B2 Receptor Gene Expression and Function

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A flow‐cytometric method for continuous measurement of intracellular Ca²⁺ concentration