Mind your assays: Misleading cytotoxicity with the WST-1 assay in the presence of manganese

Scarcello, Eleonora; Lambremont, Alexia; Vanbever, Rita; Jacques, Pascal; Lison, Dominique

doi:10.1371/journal.pone.0231634

Cited by 48 publications

(40 citation statements)

References 25 publications

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“…In this case, a known amount of hydrophilic formazan crystal formed from WST-1 should be incubated with respective MWCNT-COOH concentrations to verify whether carbon nanotubes do not interfere with the signal, yielding a false-positive result. Recently, a similar experiment with WST-1 assay was carried out by Scarcello et al on manganese nanoparticles, with the conclusion that luminescence-based tests could be more appropriate for this type of analysis [ 45 ]. This further confirms that research on the interference of nanomaterials with luminescence-based assays is needed.…”

Section: Discussionmentioning

confidence: 99%

Carbon Nanotubes Interference with Luminescence-Based Assays

et al. 2020

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Carbon nanotubes (CNTs) are one of the most promising nanomaterials synthesized to date. Thanks to their unique mechanical, electronic, and optical properties, they have found a wide application in electronics in the production of biosensors and nanocomposites. The functionalization of multiwalled carbon nanotubes (MWCNTs) is aimed at making them biocompatible by adding hydrophilic groups on their surface, increasing their solubility and thus rendering them applicable in the regenerative medicine. So far, there is conflicting information about carbon nanotubes in biological systems. This paper investigates the effect of functionalized, oxidized, multiwalled carbon nanotubes (MWCNT-Ox) on the cytotoxicity of normal human articular chondrocytes (NHAC-kn cell line). Since absorbance-based and fluorescence-based assays were shown to interfere with carbon nanotubes, luminescence-based tests were carried out, as they work on a different method of detection and provide advantages over the mentioned ones. Cell viability and reactive oxygen species (ROS) tests were carried out. The cell viability assay showed that with the increasing MWCNTs concentration, the number of viable chondrocytes was significantly decreasing. Exposure to MWCNT-Ox indicated oxidative stress in the lowest investigated concentration with a decreased amount of ROS with higher concentrations. However, control experiments with adenosine triphosphate (ATP) and H2O2—molecules that are detected by the assays—showed that carbon nanotubes interfere directly with measurement, thus rendering the results unreliable. To understand the exact interference mechanisms, further studies must be taken. In conclusion, this study shows that luminescence-based tests yield erroneous results, confirming that in vitro experiments in the literature concerning carbon nanotubes should be analyzed with caution.

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Section: Discussionmentioning

confidence: 99%

Carbon Nanotubes Interference with Luminescence-Based Assays

et al. 2020

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“…Although CS is characterized as a biocompatible material [ 15 ], cell behavior varies depending on the material characteristics and the testing system [ 43 ]. Thus, various in vitro testing methods are useful to verify if the material is potentially toxic in different ways [ 45 , 46 ]. CS particles induced the adverse cell response at high concentrations, however, this effect could be expected efficiently compensated in vivo.…”

Section: Resultsmentioning

confidence: 99%

A Facile Synthesis Process and Evaluations of α-Calcium Sulfate Hemihydrate for Bone Substitute

Nguyen

Pham³

et al. 2020

Materials

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Alpha-calcium sulfate hemihydrate (α-HH) has been used effectively in grafting through its desired features to support bone regeneration. In recent years, many synthetic methods have been proposed. Among them, the autoclave method for manufacturing α-HH is best suited for cost-savings due to its simple operation and limited use of additives. Despite these advantages, the synthesis of surgical grade products without the use of any additives has not yet been clearly discussed. In this study, surgical grade α-HH was successfully produced from calcium sulfate dihydrate (DH) using the autoclave method at an elevated temperature and pressure. The synthesized powder had a high purity of about 98.62% α-HH with a prismatic morphology (20.96 ± 8.83 µm in length and 1.30 ± 0.71 µm in diameter). The screening tests, in simulated body fluid (SBF) solution, for the product properties showed no bioactivity, and fast degradation accompanied by a slight decrease in pH. The lactate dehydrogenase (LDH) assay showed good biocompatibility of the material, however, its potential for cytotoxicity was also observed in NIH 3T3 cells. Briefly, despite some unfavorable properties, the autoclave-synthesized α-HH is a promising bone graft substitute that can be applied in orthopedic and maxillofacial surgeries.

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“…These reagents include 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2 H -Tetrazolium-5-Carboxanilide (XTT) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). One advantage of these assays is that no second reagent is required for solubilization of the formazide, allowing readings to be taken periodically [ 87 , 88 ]. Similarly, negatively charged 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) (WST-1) is reduced to another variation of water-soluble formazide.…”

Section: Examination Of Microencapsulated Islets and Cell Linesmentioning

confidence: 99%

“…Similarly, negatively charged 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) (WST-1) is reduced to another variation of water-soluble formazide. However, WST-1 is unable to penetrate the cell, so the reduction reaction occurs outside, making it unsuitable for widespread use in microencapsulated cells [ 88 , 89 ].…”

Section: Examination Of Microencapsulated Islets and Cell Linesmentioning

confidence: 99%

Advancements in Assessments of Bio-Tissue Engineering and Viable Cell Delivery Matrices Using Bile Acid-Based Pharmacological Biotechnologies

et al. 2021

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The utilisation of bioartificial organs is of significant interest to many due to their versatility in treating a wide range of disorders. Microencapsulation has a potentially significant role in such organs. In order to utilise microcapsules, accurate characterisation and analysis is required to assess their properties and suitability. Bioartificial organs or transplantable microdevices must also account for immunogenic considerations, which will be discussed in detail. One of the most characterized cases is the investigation into a bioartificial pancreas, including using microencapsulation of islets or other cells, and will be the focus subject of this review. Overall, this review will discuss the traditional and modern technologies which are necessary for the characterisation of properties for transplantable microdevices or organs, summarizing analysis of the microcapsule itself, cells and finally a working organ. Furthermore, immunogenic considerations of such organs are another important aspect which is addressed within this review. The various techniques, methodologies, advantages, and disadvantages will all be discussed. Hence, the purpose of this review is providing an updated examination of all processes for the analysis of a working, biocompatible artificial organ.

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Mind your assays: Misleading cytotoxicity with the WST-1 assay in the presence of manganese

Cited by 48 publications

References 25 publications

Carbon Nanotubes Interference with Luminescence-Based Assays

Carbon Nanotubes Interference with Luminescence-Based Assays

A Facile Synthesis Process and Evaluations of α-Calcium Sulfate Hemihydrate for Bone Substitute

Advancements in Assessments of Bio-Tissue Engineering and Viable Cell Delivery Matrices Using Bile Acid-Based Pharmacological Biotechnologies

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