Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells

Stapelfeldt, Karsten; Ehrke, Eric; Steinmeier, Johann; Rastedt, Wiebke; Dringen, Ralf

doi:10.1016/j.ab.2017.09.011

Cited by 22 publications

(22 citation statements)

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“…10). As shown for menadione [46], also the β-lap/β-lapachol redox pair was found to efficiently act as electron cycler and to mediate the electron transfer from cellular sources for extracellular WST1 reduction which requires export of β-lapachol and reuptake of β-lap ( Fig. 10).…”

Section: Discussionmentioning

confidence: 75%

“…The water-soluble tetrazolium salt 1 (WST1) is a membrane impermeable substance that can be reduced in cell cultures by membrane permeable electron cyclers to form a water-soluble formazan product [ 46 ]. To test whether β-lap can serve as electron cycler for WST1 reduction, cultured astrocytes were washed once with 1 mL of IB and then incubated in 200 µL of IB containing 5 mM glucose, 20 µM β-lap and 400 µM WST1 in the absence or the presence of other compounds which are listed in the legend of Fig.…”

Section: Methodsmentioning

confidence: 99%

“…After 10 min or 30 min of incubation, 50 µL of incubation solution were harvested, diluted with 150 µL of water in wells of a microtiter plate and the absorbance of the WST1 formazan generated was measured at 450 nm in a plate reader. The concentration of WST1 formazan in the incubation medium was calculated from the absorbance by using the extinction coefficient of 35.2 mM −1 × cm −1 [46].…”

Section: Determination Of Extracellular Wst1 Formazan Productionmentioning

confidence: 99%

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β-Lapachone Induces Acute Oxidative Stress in Rat Primary Astrocyte Cultures that is Terminated by the NQO1-Inhibitor Dicoumarol

et al. 2020

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β-lapachone (β-lap) is reduced in tumor cells by the enzyme NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1) to a labile hydroquinone which spontaneously reoxidises to β-lap, thereby generating reactive oxygen species (ROS) and oxidative stress. To test for the consequences of an acute exposure of brain cells to β-lap, cultured primary rat astrocytes were incubated with β-lap for up to 4 h. The presence of β-lap in concentrations of up to 10 µM had no detectable adverse consequences, while higher concentrations of β-lap compromised the cell viability and the metabolism of astrocytes in a concentration- and time-dependent manner with half-maximal effects observed for around 15 µM β-lap after a 4 h incubation. Exposure of astrocytes to β-lap caused already within 5 min a severe increase in the cellular production of ROS as well as a rapid oxidation of glutathione (GSH) to glutathione disulfide (GSSG). The transient cellular accumulation of GSSG was followed by GSSG export. The β-lap-induced ROS production and GSSG accumulation were completely prevented in the presence of the NQO1 inhibitor dicoumarol. In addition, application of dicoumarol to β-lap-exposed astrocytes caused rapid regeneration of the normal high cellular GSH to GSSG ratio. These results demonstrate that application of β-lap to cultured astrocytes causes acute oxidative stress that depends on the activity of NQO1. The sequential application of β-lap and dicoumarol to rapidly induce and terminate oxidative stress, respectively, is a suitable experimental paradigm to study consequences of a defined period of acute oxidative stress in NQO1-expressing cells.

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Section: Discussionmentioning

confidence: 75%

Section: Methodsmentioning

confidence: 99%

Section: Determination Of Extracellular Wst1 Formazan Productionmentioning

confidence: 99%

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β-Lapachone Induces Acute Oxidative Stress in Rat Primary Astrocyte Cultures that is Terminated by the NQO1-Inhibitor Dicoumarol

et al. 2020

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“…Mechanisms of DNA repair such as nucleotide-excision, homologous recombination and non-homologous end-joining [ 26 , 27 ] all require DNA synthesis [ 28 ] and would be labelled by BrdU incorporation. While cell-cycle duration may vary between different types of cells, additional assays of proliferation (e.g., Ki67 or flow-cytometry studies using BrdU and propidium iodide [ 29 ]) and metabolic activity (e.g., cell reduction of tetrazolium salts such as MTT and WST1 [ 30 , 31 ]) would complement findings on surviving-cell density and further account for BrdU labeling of DNA synthesis in relation to cell cycling and/or DNA repair during apoptosis.…”

Section: Discussionmentioning

confidence: 99%

A mechanism for semaphorin-induced apoptosis: DNA damage of endothelial and myogenic cells in primary cultures from skeletal muscle

2018

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One hallmark of cancer is its ability to recruit a vascular supply to support rapid growth. Suppression of angiogenesis holds potential as a second-line or adjuvant therapy to stunt cancer growth, progression, metastasis, and post-resection regeneration. To begin to test the hypothesis that semaphorin 3A and 3F together, will induce endothelial cell apoptosis by inducing DNA damage, mixed primary cultures isolated from normal adult mouse skeletal muscle were treated for 48 hr with Sema3A ± Sema3F (100ng/mL). Changes in surviving-cell density, DNA synthesis, DNA repair (gamma-Histone 2AX, γH2AX, an indirect measure for DNA damage), and apoptotic DNA fragmentation (TUNEL staining) were assayed in cultures of CD31+ endothelial and desmin+ muscle cells. Sema3F increased DNA damage-associated DNA repair in both cell types. Co-treatment with Sema3A+3F increased γH2AX staining ~25-fold over control levels, and further increased apoptosis compared to control and Sema3A alone. Results were negated by treatment with neutralizing anti-semaphorin antibodies and are interpreted as suggesting that Sema3A may sensitize endothelial but not muscle cells to Sema3F-induced DNA damage. These preliminary findings on a complex system of interacting cells may contribute to developing applications that could target angiogenic regulatory mechanisms for their therapeutic potential against cancer progression and metastasis.

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“…The C6 glioma cell line was kindly provided by Dr. Frank Dietz (University of Bremen). These cells express the astrocytic marker protein glial-fibrillary acidic protein [51] and were cultured as described earlier [29]. From a 175 cm 2 cell-culture flask, 80% confluent cultures were harvested and were seeded in 1 mL culture medium (90% DMEM containing 25 mM d-glucose, 1 mM sodium pyruvate, 18 U/ mL penicillin G, 18 µg/mL streptomycin sulphate and 10% FCS; pH 7.4) at a density of 200,000 viable cells per mL into wells of 24-well culture plates.…”

Section: Cell Culture and Experimental Incubationsmentioning

confidence: 99%

Iron-Doping of Copper Oxide Nanoparticles Lowers Their Toxic Potential on C6 Glioma Cells

et al. 2020

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Copper oxide nanoparticles (CuO-NPs) are well known for their cytotoxicity which in part has been attributed to the release of copper ions from CuO-NPs. As iron-doping has been reported to reduce the susceptibility of CuO-NPs to dissolution, we have compared pure CuO-NPs and CuO-NPs that had been doped with 10% iron (CuO-Fe-NPs) for copper release and for their toxic potential on C6 glioma cells. Physicochemical characterization revealed that dimercaptosuccinate (DMSA)-coated CuO-NPs and CuO-Fe-NPs did not differ in their size or zeta potential. However, the redox activity and liberation of copper ions from CuO-Fe-NPs was substantially slower compared to that from CuO-NPs, as demonstrated by cyclic voltammetry and by the photometric quantification of the copper ion-bathocuproine complex, respectively. Exposure of C6 cells to these NPs caused an almost identical cellular copper accumulation and each of the two types of NPs induced ROS production and cell toxicity. However, the time-and concentration-dependent loss in cell viability was more severe for cells that had been treated with CuO-NPs compared to cells exposed to CuO-Fe-NPs. Copper accumulation and toxicity after exposure to either CuO-NPs or CuO-Fe-NPs was prevented in the presence of copper chelators, while neutralization of the lysosomal pH by bafilomycin A1 prevented toxicity without affecting cellular copper accumulation or ROS production. These data demonstrate that iron-doping does not affect cellular accumulation of CuO-NPs and suggests that the intracellular liberation of copper ions from CuO-NPs is slowed by the iron doping, which in turn lowers the cell toxic potential of iron-doped CuO-NPs.

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Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells

Cited by 22 publications

References 20 publications

β-Lapachone Induces Acute Oxidative Stress in Rat Primary Astrocyte Cultures that is Terminated by the NQO1-Inhibitor Dicoumarol

β-Lapachone Induces Acute Oxidative Stress in Rat Primary Astrocyte Cultures that is Terminated by the NQO1-Inhibitor Dicoumarol

A mechanism for semaphorin-induced apoptosis: DNA damage of endothelial and myogenic cells in primary cultures from skeletal muscle

Iron-Doping of Copper Oxide Nanoparticles Lowers Their Toxic Potential on C6 Glioma Cells

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