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BMC Research Notes Feb 2015The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a... (Comparative Study)
Comparative Study
BACKGROUND
The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration assays; the neutral red uptake (NRU), resazurin reduction (RES) and sulforhodamine B (SRB) assays, in comparison to the MTT assay. Interference between the MTT assay and three glycolysis inhibitors, 2-deoxyglucose, 3-bromopyruvate and lonidamine, was investigated.
RESULTS
Data indicate that the NRU, RES and SRB assays showed the smallest variability across the linear range, while the largest variation was observed for the MTT assay. This implies that these assays would more accurately detect small changes in cell number than the MTT assay. The SRB assay provided the most reproducible results as indicated by the coefficient of determination after a limited number of experiments. The SRB assay also produced the lowest variance in the derived 50% inhibitory concentration (IC50), while IC50 concentrations of 3-bromopyruvate could not be detected using either the MTT or RES assays after 24 hours incubation. Interference in the MTT assay was observed for all three tested glycolysis inhibitors in a cell-free environment. No interferences were observed for the NRU, SRB or RES assays.
CONCLUSIONS
This study demonstrated that the MTT assay was not the best assay in a number of parameters that must be considered when a cell enumeration assay is selected: the MTT assay was less accurate in detecting changes in cell number as indicated by the variation observed in the linear range, had the highest variation when the IC50 concentrations of the glycolysis inhibitors were determined, and interference between the MTT assay and all the glycolysis inhibitors tested were observed. The SRB assay performed best overall considering all of the parameters, suggesting that it is the most suitable assay for use in preclinical screening of novel therapeutic compounds with oxido-reductive potential.
Topics: Artifacts; Biological Assay; Cell Count; Cell Line, Tumor; Cell Survival; Deoxyglucose; Glycolysis; Humans; Indazoles; Neutral Red; Observer Variation; Oxazines; Oxidation-Reduction; Pyruvates; Reproducibility of Results; Rhodamines; Sensitivity and Specificity; Staining and Labeling; Tetrazolium Salts; Thiazoles; Xanthenes
PubMed: 25884200
DOI: 10.1186/s13104-015-1000-8 -
Yakugaku Zasshi : Journal of the... 2021Phototoxicity is a toxic response elicited by topically applied or systemically administered photoreactive chemicals after exposure to light and can be broadly... (Review)
Review
Phototoxicity is a toxic response elicited by topically applied or systemically administered photoreactive chemicals after exposure to light and can be broadly categorized into photoirritation, photoallergy, photogenotoxicity, and photocarcinogenicity. The need in the 21st century for accurate evaluation of photosafety has led to the publication of a number of guidelines from government agencies in Europe and the U.S.A. as well as the Organisation for Economic Co-operation and Development (OECD). In this review, we first discuss the mechanisms of phototoxicity and how they can be evaluated. We then discuss the state of the art and challenges now faced in photosafety evaluation of pharmaceuticals and cosmetics. Additionally, we describe the latest developments in OECD test guidelines (TG) for assessing photosafety, including revisions to the in vitro 3T3 neutral red uptake (NRU) phototoxicity test (TG 432) and the newly adopted reactive oxigen species (ROS) assay (TG 495). We will emphasize the importance of selecting the most appropriate means of evaluation with reference to the latest guidelines and other legal criteria for conducting photosafety evaluation.
Topics: 3T3 Cells; Animals; Cells, Cultured; Dermatitis, Phototoxic; Humans; Light; Mice; Neutral Red; Reactive Oxygen Species; Safety; Toxicity Tests
PubMed: 33390438
DOI: 10.1248/yakushi.20-00148 -
Heliyon Jul 2021Research indicates the use of adsorbent materials to remove pollutants from wastewater and effluents, which can be obtained from renewable materials such as biomass,...
Research indicates the use of adsorbent materials to remove pollutants from wastewater and effluents, which can be obtained from renewable materials such as biomass, biopolymers (chitosan) or composites. Thus, the objective of this work was to produce and evaluate activated carbon (AC) and chitosan composite films as adsorbents of neutral red dye. AC films were produced using CO and water vapor. The variables of the activation process were time (1 and 2 h) and temperature (600 and 750 °C). Five films were produced, with one pure chitosan (T1) film and four activated carbon with chitosan films (T2, T3, T4 and T5). The T2 film refers to activated carbon produced at 600 °C for 1 h + chitosan, T3 to activated carbon produced at 600 °C for 2 h + chitosan, T4 to activated carbon produced at 750 °C for 1 h + chitosan and T5 to activated carbon produced at 750 °C for 2 h + chitosan. The T5 film increased its adsorption capacity by approximately 87% and its removal efficiency of neutral red dye by 43% compared to T1. The presence of activated carbon in the films provided an increase in the adsorption capacity of the neutral red dye.
PubMed: 34381895
DOI: 10.1016/j.heliyon.2021.e07629 -
Analytical and Bioanalytical Chemistry Jul 2022Carbon nanodots modified with Neutral Red covalently inserted in the nanostructure (NR-CDs) have been prepared by a simple synthesis method based on microwave...
Carbon nanodots modified with Neutral Red covalently inserted in the nanostructure (NR-CDs) have been prepared by a simple synthesis method based on microwave irradiation under controlled temperature and pressure. The synthetized NR-CDs have been characterized by different techniques, demonstrating the covalent bonding of Neutral Red molecules to the carbon dots nanostructure. Fluorescence activity of the prepare NR-CDs has been explored showing different interaction pathways with singled and doubled stranded DNA. These studies have been successfully applied to develop a new fluorescence DNA hybridization assay to the detection of a specific DNA sequence of Escherichia coli bacteria.
Topics: Carbon; DNA; Fluorescent Dyes; Neutral Red; Quantum Dots; Spectrometry, Fluorescence
PubMed: 35288763
DOI: 10.1007/s00216-022-03980-1 -
Bioresource Technology Nov 2015The aim of this work was to compare the effects of electrosynthesis on different bacterial species. The effects of neutral red-mediated electrosynthesis on the...
The aim of this work was to compare the effects of electrosynthesis on different bacterial species. The effects of neutral red-mediated electrosynthesis on the metabolite profiles of three microorganisms: Escherichia coli, Klebsiella pneumoniae, and Zymomonas mobilis, were measured and compared and contrasted. A statistically comprehensive analysis of neutral red-mediated electrosynthesis is presented using the analysis of end-product profiles, current delivered, and changes in cellular protein expression. K. pneumoniae displayed the most dramatic response to electrosynthesis of the three bacteria, producing 93% more ethanol and 76% more lactate vs. control fermentation with no neutral red and no electron delivery. Z. mobilis showed no response to electrosynthesis except elevated acetate titers. Stoichiometric comparison showed that NAD(+) reduction by neutral red could not account for changes in metabolites during electrosynthesis. Neutral red-mediated electrosynthesis was shown to have multifarious effects on the three species.
Topics: Batch Cell Culture Techniques; Bioelectric Energy Sources; Electrochemical Techniques; Electrons; Escherichia coli; Fermentation; Klebsiella pneumoniae; Neutral Red; Zymomonas
PubMed: 26096579
DOI: 10.1016/j.biortech.2015.06.005 -
Current Research in Microbial Sciences Dec 2021Microbial fuel cells (MFCs) that generate bioelectricity from biodegradable waste have received considerable attention from biologists. Fungi play a significant role as... (Review)
Review
Microbial fuel cells (MFCs) that generate bioelectricity from biodegradable waste have received considerable attention from biologists. Fungi play a significant role as both anodic and cathodic catalysts in MFCs. is a fungus with an ability to transfer electrons through mediators such as methylene blue (MB), neutral red (NR) or even without a mediator. This unique role of fungal cells in exocellular electron transfer (EET) and their interactions with electrodes hold a lot of promise in areas such as wastewater treatment where yeast cell-based MFCs can be used. The present article highlights the physico-chemical factors affecting the performance of fungal-mediated MFCs in terms of power output and degradation of organic pollutants, along with the challenges associated with fungal MFCs. In addition, to this comparative assessment of fungal-mediated bio-electrochemical systems, their development, possible applications and potential challenges are also discussed.
PubMed: 34841332
DOI: 10.1016/j.crmicr.2021.100041 -
Biotechnic & Histochemistry : Official... May 2021Harsh conditions within the tumor microenvironment, such as hypoxia and extracellular acidic pH (pH), inactivate some chemotherapies, which results in limited or no...
Harsh conditions within the tumor microenvironment, such as hypoxia and extracellular acidic pH (pH), inactivate some chemotherapies, which results in limited or no cytotoxicity. Standard MTT, ATPlite and protease assays that are used to determine the potency of newly developed drugs often give erroneous results when applied under hypoxic or acidic conditions. Therefore, development of a cytotoxicity assay that does not yield false positive or false negative results under circumstances of both hypoxia and acidic pH is needed. We evaluated currently used cell viability assays as well as neutral red staining to assess viability of ovarian and pancreatic cancer cells grown in an acidic pH microenvironment after treatment with carboplatin, gemcitabine or chloroquine. We validated cell viability using western blotting of pro-caspase-9 and cleaved-caspase-9, and LC3-I and - II. Standard cell viability assays indicated cell viability accurately at pH 7.4, but was not correlated with induction of apoptosis or autophagy at acidic pH. By contrast, our modified neutral red assay detected cell viability accurately over a range of pH as demonstrated by its correlation with induction of apoptosis and autophagy. Neutral red staining is effective for evaluating the effect of chemotherapeutic agents on cell viability under acidic pH or hypoxic conditions.
Topics: Antineoplastic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Cell Survival; Humans; Neutral Red
PubMed: 32744455
DOI: 10.1080/10520295.2020.1802065 -
BMC Cancer Oct 2021Cancer remains one of the leading causes of death worldwide, despite the possibilities to detect early onset of the most common cancer types. The search for the optimal...
BACKGROUND
Cancer remains one of the leading causes of death worldwide, despite the possibilities to detect early onset of the most common cancer types. The search for the optimal therapy is complicated by the cancer diversity within tumors and the unsynchronized development of cancerous cells. Therefore, it is necessary to characterize cancer cell populations after treatment has been applied, because cancer recurrence is not rare. In our research, we concentrated on small cancer cell subpopulation (microcells) that has a potential to be cancer resistance source. Previously made experiments has shown that these cells in small numbers form in specific circumstances after anticancer treatment.
METHODS
In experiments described in this research, the anticancer agents' paclitaxel and doxorubicin were used to stimulate the induction of microcells in fibroblast, cervix adenocarcinoma, and melanoma cell lines. Mainly for the formation of microcells in melanoma cells. The drug-stimulated cells were then characterized in terms of their formation efficiency, morphology, and metabolic activity.
RESULTS
We observed the development of cancer microcells and green fluorescent protein (GFP) transfection efficiency after stress. In the time-lapse experiment, we observed microcell formation through a renewal process and GFP expression in the microcells. Additionally, the microcells were viable after anticancer treatment, as indicated by the nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) enzyme activity assay results. Taken together, these findings indicate that cancer microcells are viable and capable of resisting the stress induced by anticancer drugs, and these cells are prone to chemical substance uptake from the environment.
CONCLUSION
Microcells are not only common to a specific cancer type, but can be found in any tumor type. This study could help to understand cancer emergence and recurrence. The appearance of microcells in the studied cancer cell population could be an indicator of the individual anticancer therapy effectiveness and patient survival.
Topics: Adenocarcinoma; Antineoplastic Agents; Cell Count; Cell Line, Tumor; Cell Nucleus; Cell Self Renewal; Cell Survival; DNA-Binding Proteins; Doxorubicin; Drug Resistance, Neoplasm; Endosomal Sorting Complexes Required for Transport; Female; Fibroblasts; Green Fluorescent Proteins; HeLa Cells; Humans; Indicators and Reagents; Melanoma; Microscopy, Electron; NADP; Neoplasm Recurrence, Local; Neoplasms; Neutral Red; Paclitaxel; Stress, Physiological; Time-Lapse Imaging; Transcription Factors; Transfection; Uterine Cervical Neoplasms
PubMed: 34625031
DOI: 10.1186/s12885-021-08813-5 -
Scientific Reports Jun 2023Assessing the in vitro toxicity of compounds on cell cultures is an important step during the screening of candidate molecules for diverse applications. Among the...
Assessing the in vitro toxicity of compounds on cell cultures is an important step during the screening of candidate molecules for diverse applications. Among the strategies employed to determine cytotoxicity, MTT, neutral red, and resazurin are commonly used. Methylene blue (MB), a phenothiazinium salt, has several uses, such as dye, redox indicator, and even as treatment for human disease and health conditions, such as malaria and methemoglobinemia. However, MB has only been sparsely used as a cellular toxicity indicator. As a viability indicator, MB is mostly applied to fixed cultures at high concentrations, especially when compared to MTT or neutral red. Here we show that MB and its related compounds new methylene blue (NMB), toluidine blue O (TBO), and dimethylmethylene blue (DMMB) can be used as cytotoxicity indicators in live (non-fixed) cells treated for 72 h with DMSO and cisplatin. We compared dye uptake between phenothiazinium dyes and neutral red by analyzing supernatant and cell content via visible spectra scanning and microscopy. All dyes showed a similar ability to assess cell toxicity compared to either MTT or neutral red. Our method represents a cost-effective alternative to in vitro cytotoxicity assays using cisplatin or DMSO, indicating the potential of phenothiazinium dyes for the screening of candidate drugs and other applications.
Topics: Humans; Coloring Agents; Phenothiazines; Cisplatin; Neutral Red; Dimethyl Sulfoxide; Methylene Blue
PubMed: 37353536
DOI: 10.1038/s41598-023-36721-0 -
ACS Nano Nov 2023Carbon dots are carbon-based nanoparticles renowned for their intense light-emitting capabilities covering the whole visible light range. Achieving carbon dots emitting...
Carbon dots are carbon-based nanoparticles renowned for their intense light-emitting capabilities covering the whole visible light range. Achieving carbon dots emitting in the red region with high efficiency is extremely relevant due to their huge potential in biological applications and in optoelectronics. Currently, photoluminescence in such an energy interval is often associated with polyheterocyclic molecular domains forming during the synthesis that, however, present low emission efficiency and issues in controlling the optical features. Here, we overcome these problems by solvothermally synthesizing carbon dots starting from Neutral Red, a common red-emitting dye, as a molecular precursor. As a result of the synthesis, such molecular fluorophore is incorporated into a carbonaceous core while retaining its original optical properties. The obtained nanoparticles are highly luminescent in the red region, with a quantum yield comparable to that of the starting dye. Most importantly, the nanoparticle carbogenic matrix protects the Neutral Red molecules from photobleaching under ultraviolet excitation while preventing aggregation-induced quenching, thus allowing solid-state emission. These advantages have been exploited to develop a fluorescence-based color conversion layer by fabricating polymer-based highly concentrated solid-state carbon dot nanocomposites. Finally, the dye-based carbon dots demonstrate both stable Fabry-Perot lasing and efficient random lasing emission in the red region.
PubMed: 37870465
DOI: 10.1021/acsnano.3c05566