Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay

Silva, Filomena S. G.; Starostina, I. G.; Ivanova, Vilena V.; Rizvanov, Albert A.; Oliveira, Paulo J.; Pereira, Susana P.

doi:10.1002/cptx.1

Cited by 41 publications

(34 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The cytotoxicity profile of the compounds under study was evaluated in differentiated SH-SY5Y cells (8,000 cells/well), according the differentiation protocol previously described, and in HepG2 cells (2.5 × 10 4 cells/well), seeded into 96-well plates. Then, the cells were exposed to increased concentrations of the test compounds (1, 10 and 50 μM) in cell culture medium for 24 h in SH-SY5Y cells or 48 h in HepG2 cells and the cytotoxicity was evaluated through measuring changes in cellular metabolic activity using the MTT or resazurin reduction assays, respectively (Silva et al, 2016 ). In both assays, the reduction of MTT tetrazolium salt or resazurin to MTT formazan or resorufin, respectively, by cellular dehydrogenases present in viable cells, gives insights on cell metabolic activity.…”

Section: Methodsmentioning

confidence: 99%

Hydroxybenzoic Acid Derivatives as Dual-Target Ligands: Mitochondriotropic Antioxidants and Cholinesterase Inhibitors

et al. 2018

Self Cite

View full text Add to dashboard Cite

Alzheimer's disease (AD) is a multifactorial age-related disease associated with oxidative stress (OS) and impaired cholinergic transmission. Accordingly, targeting mitochondrial OS and restoring cholinergic transmission can be an effective therapeutic strategy toward AD. Herein, we report for the first time dual-target hydroxybenzoic acid (HBAc) derivatives acting as mitochondriotropic antioxidants and cholinesterase (ChE) inhibitors. The studies were performed with two mitochondriotropic antioxidants AntiOxBEN1 (catechol derivative), and AntiOxBEN2 (pyrogallol derivative) and compounds 15–18, which have longer spacers. Compounds AntiOxBEN1 and 15, with a shorter carbon chain spacer (six- and eight-carbon) were shown to be potent antioxidants and BChE inhibitors (IC50 = 85 ± 5 and 106 ± 5 nM, respectively), while compounds 17 and 18 with a 10-carbon chain were more effective AChE inhibitors (IC50 = 7.7 ± 0.4 and 7.2 ± 0.5 μM, respectively). Interestingly, molecular modeling data pointed toward bifunctional ChEs inhibitors. The most promising ChE inhibitors acted by a non-competitive mechanism. In general, with exception of compounds 15 and 17, no cytotoxic effects were observed in differentiated human neuroblastoma (SH-SY5Y) and human hepatocarcinoma (HepG2) cells, while Aβ-induced cytotoxicity was significantly prevented by the new dual-target HBAc derivatives. Overall, due to its BChE selectivity, favorable toxicological profile, neuroprotective activity and drug-like properties, which suggested blood-brain barrier (BBB) permeability, the mitochondriotropic antioxidant AntiOxBEN1 is considered a valid lead candidate for the development of dual acting drugs for AD and other mitochondrial OS-related diseases.

show abstract

Section: Methodsmentioning

confidence: 99%

Hydroxybenzoic Acid Derivatives as Dual-Target Ligands: Mitochondriotropic Antioxidants and Cholinesterase Inhibitors

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…Dye bound to cell proteins was extracted with 10 mM Tris-base solution, pH 10. After SRB labeling, absorbance was measured in a Biotek Cytation 3 spectrophotometer at 510 nm with background correction at 620 nm, the amount of dye released is proportional to the number of cell mass in each well, as demonstrated previously (Silva et al, 2016;Vichai and Kirtikara, 2006).…”

Section: Cell Proliferation Measurementmentioning

confidence: 99%

“…After removing the PBS 1Â, 500 ll of resazurin solution (1:1000 dilution in growth medium from a stock solution) was added in each well and incubated for 6 h at 37 C with a 5% CO 2 atmosphere. Resorufin fluorescence was measured in a Biotek Cytation 3 spectrophotometer using excitation wavelength of 540 nm and emission of 590 nm (Silva et al, 2016).…”

Section: Metabolic Activity Determinationmentioning

confidence: 99%

Metabolic and Phenotypic Characterization of Human Skin Fibroblasts After Forcing Oxidative Capacity

Pereira

Deus

Serafim

et al. 2018

Toxicological Sciences

View full text Add to dashboard Cite

Human skin fibroblasts present technical advantages for the study of mitochondrial-induced toxicity, because those cells can be isolated from patients by lowly invasive methods and present specific cumulative cellular damage and mutations of particular conditions. Several drugs lead to organ toxicity, with some of these drugs having been already withdrawn from the market. Frequently, drug-induced toxicity is attributed to mitochondrial liabilities. One of the approaches to identify drug-induced mitochondrial toxicity is using glucose-free/galactose/glutamine/pyruvate-containing cell culture media that force cells to be more dependent on oxidative phosphorylation for energy production. However, the effects of this modified culture medium itself on the mitochondrial phenotype of human skin fibroblasts have not been explored in detail. Our objective was to assess the mitochondrial biology of human skin fibroblasts under standard or modified culture conditions so that system can be validated and used in a more reliable way to disclose mitochondrial liabilities of drug candidates or intrinsic metabolic differences in fibroblasts. Our results showed that forcing mitochondrial remodeling in human skin fibroblasts increased oxygen consumption rate, ATP levels, and mitochondria-related transcripts and proteins. Moreover, the metabolic remodeling increased cytotoxicity of mitochondrial poisons. In general, no alterations in gene expression related with differentiation status were observed in human skin fibroblasts, with exception of increased paxilin gene expression. Not only the current work highlights the importance of using human skin primary cells to study drug-induced mitochondrial toxicity, it also reinforces the use of this tool to detect specific mitochondrial defects in skin fibroblasts from patients.

show abstract

“…After removing the PBS 1×, 100 μL of resazurin solution (1:100 dilution in growth medium from a stock solution) was added to each well and incubated for 4 h at 37°C with a 5% CO 2 atmosphere. Resorufin fluorescence was measured in a BioTek Cytation 3 spectrophotometer using excitation wavelength of 540 nm and emission of 590 nm (Silva et al 2016). The spectral characteristics of DMEM and resazurin and menadione (25 µM) were determined in 96-well plate by BioTek Cytation 3 spectrophotometer using wavelength between 350 and 700 nm (visible spectrum).…”

Section: Journal Of Molecular Endocrinologymentioning

confidence: 99%

SULFATION PATHWAYS: Potential benefits of a sulfated resveratrol derivative for topical application

Correia-da-Silva

Rocha

Marques

et al. 2018

Journal of Molecular Endocrinology

View full text Add to dashboard Cite

Resveratrol (RSV) is a polyphenolic compound with antioxidant, anti-inflammatory and anti-aging properties partly associated with sirtuin 1 (SIRT1)-activation in the skin. However, poor water solubility may limit RSV efficacy. This work aimed to clarify the interest of a new synthetic water-soluble RSV derivative (resveratrol glucoside sulfate, RSV-GS) for topical application. Resveratrol glucoside sulfate was synthesized using microwave-assisted sulfation. Cytotoxicity assays were performed with the keratinocyte HaCaT cell line, using MTT reduction, neutral red uptake, Alamar Blue/resazurin reduction, trypan blue exclusion and measurement of ATP concentration. Western blotting was used to evaluate SIRT1 protein content. Regarding SIRT1 binding, an docking study was performed, using AutoDock Vina. Our results showed that the synthetic derivative RSV-GS was 1000 times more soluble in water than RSV and its non-sulfated glucoside. No relevant decrease in HaCaT cell viability was observed for concentrations up to 5 mM for RSV-GS, and up to 500 μM for resveratrol glucoside, while a significant decrease in HaCaT viability occurred from 100 μM for RSV. RSV-GS and RSV showed a similar behavior regarding protective effect against oxidative stress-induced cytotoxicity. SIRT1 protein content increased after treatment with 500 μM of RSV-GS and 100 μM of RSV. Moreover, studies predicted that RSV-GS binds more stably to SIRT1 with a lower binding free energy than RSV. Although these results support the possible use of RSV-GS in topical formulations, safety and efficacy studies are needed before considering the use of RSV-GS in commercial products.

show abstract

Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay

Cited by 41 publications

References 44 publications

Hydroxybenzoic Acid Derivatives as Dual-Target Ligands: Mitochondriotropic Antioxidants and Cholinesterase Inhibitors

Hydroxybenzoic Acid Derivatives as Dual-Target Ligands: Mitochondriotropic Antioxidants and Cholinesterase Inhibitors

Metabolic and Phenotypic Characterization of Human Skin Fibroblasts After Forcing Oxidative Capacity

SULFATION PATHWAYS: Potential benefits of a sulfated resveratrol derivative for topical application

Contact Info

Product

Resources

About