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Cellular and Molecular Toxicology of Nanoparticles
Published in Vladimir Torchilin, Handbook of Materials for Nanomedicine, 2020
A. Zielińska, D. Santos, J. R. Campos, A. Santini, P. Severino, A. A. M. Shimojo, S. B. Souto, E. B. Souto
The rate of cellular metabolism can be estimated by assessing the activity of mitochondrial enzymes. This is done mainly by colorimetric tests based on the Triazolic dyes [58]. In normal conditions, these dyes suffer mitochondrial reduction by enzymatic cleavage. The tetrazolium bromide test ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) (MTT) consists of a reduction of the tetrazole ring. The dye is cleaved and transformed into a blue/purple compound, formazan (E,Z-1-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan), which is an insoluble salt. This reaction only occurs in metabolically viable cells by the mitochondrial enzymes. The amount of formazan formed reflects the rate of cellular metabolism and its amount is proportional to the number of viable cells. The quantification of the formazan formed is done after solubilization and standardization using spectrophotometry [22, 55, 58].
Nanofiber Electrospun Membrane Based on Biodegradable Polymers for Biomedical and Tissue Engineering Application
Published in Ahmad Fauzi Ismail, Nidal Hilal, Juhana Jaafar, Chris J. Wright, Nanofiber Membranes for Medical, Environmental, and Energy Applications, 2019
Lim Mim Mim, Naznin Sultana, Hasrinah Hasbullah, Madzlan Aziz
Cell attachment and proliferation can be observed by determining the number of cells on the scaffold. Counting cell number after cells are trypsinized is the most straightforward method for cell attachment and proliferation. However, some cells are difficult to be detached especially from a rough surface. There are some colorimetric methods to determine the number of cells including the MTT assay and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Both these assays measure the cellular metabolic activity of cells. Viable cells contain dehydrogenase enzymes which will reduce the tetrazolium salt of MTT and MTS to purple formazan. The formazan can be dissolved in dimethyl sulfoxide and quantified by optical absorbance. The absorbance reflects the number of viable cells. The advantage of using MTT and MTS methods is cells do not need to be detached from the scaffold. However, it also has a disadvantage that different cells contain different amount of dehydrogenase enzyme (Ma et al. 2007). Several studies have used MTT and MTS for cell attachment and proliferation studies (Ndreu 2007; Kumbar et al. 2008; Sultana and Wang 2012). In addition, DAPI staining can also be used to investigate cell attachment on scaffolds.
Applications of Liquid Marbles
Published in Andrew Terhemen Tyowua, Liquid Marbles, 2018
Following incubation, the viability of cells in the marbles containing Fe3+ was measured by color change using AlmarBlue reagent or MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) colorimetric assay or DNA quantification and compared with those without the metal ion. In AlmarBlue colorimetric assay, a resazurin-based product (blue) is reduced to resofurin (red) in the presence of viable cells. Similarly, in the MTS assay, a tetrazolium salt is reduced to a formazan product with an associated color change of yellow to brown in the presence of viable cells. The intensity of the resultant color and DNA amount ratio (as compared with a marble containing phosphate buffer saline) was seen to decrease with increasing Fe3+ concentration as expected, indicative that cell viability decreases with Fe3+ concentration and that Fe3+ toxicity is concentration dependent. This shows that liquid marbles can be used for high-throughput drug screening of anchorage-dependent cells as well as other cell types.
Implications of toxicity testing for health risk assessment of vapor-phase and PM2.5-bound polycyclic aromatic hydrocarbons during the diesel engine combustion
Published in Human and Ecological Risk Assessment: An International Journal, 2022
Guan-Fu Chen, Ying-Chi Lin, Yuan-Chung Lin, Chia-Chi Wang, Wei-Hsiang Chen
The MTT assay is a colorimetric non-clonogenic method and has been widely used to analyze cell viability (Shen et al. 2011; Cheng et al. 2017). Briefly, one hundred ml of cells (5 × 104 cell/ml) were seeded into 96-well plates (Sigma-Aldrich, USA) at 37 °C and incubated in a chamber environment of 5% CO2. After adding sample extracts with different concentrations, cells were incubated for 24 h. The supernatants were removed and phosphate buffer saline (PBS) was used to wash the cells twice. The tetrazolium salt (MTT; 2 mg/ml in PBS) was added to each well (100 µl/well). The cells were incubated in a chamber environment of 5% CO2 at 37 °C for 4 h. Nicotinamide adenine dinucleotide (NADH) was used as the substrate in the assay. The tetrazolium salt was reduced by actively growing cells to produce a purple insoluble formazan product. The MTT reducing activity was proportional to the accumulation of purple coloration indicative of the cell metabolization and proliferation. The formed formazan was dissolved in a lysis buffer (DMSO) overnight in the dark. After colorization and color homogenization, the optical density of cells was measured between 570 and 650 nm by using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Dynatech Lab. Inc., USA). The positive control was the cells treated with 10% DMSO (Bae et al. 2012). Blank tests were carried out by adding isopropanol (200 µL) into each well and following the similar steps described above.
Preparation and modeling of electrospun polyhydroxybutyrate/polyaniline composite scaffold modified by plasma and printed by an inkjet method and its cellular study
Published in Journal of Biomaterials Science, Polymer Edition, 2020
Mohammad Zamanifard, Mohammad Taghi Khorasani, Morteza Daliri, Mahmoud Parvazinia
Survival and cell proliferation assay were performed using L929 and HDF cells lines in accordancewith ISO10993-5. Cells were cultured with DMEM medium containing 100 IU/ml penicillin and 100 µg/ml streptomycin, supplemented with 10% FBS. A cell suspension with a concentration of 1 × 104/ml was prepared. Scaffold samples with 1 cm diameters were sterilized in cell culture hood under the UV light (390 wavelengths) with a distance of 20 to 30 cm with an exposure time of 20 min for each side. Scaffolds were placed in 24 well plates inculture medium for one day before cell seeding. This was done to make sure that the samples were not contaminated and also to facilitate the absorption of the cells. The scaffolds were transferred to 24 well plates, and 1 ml cell suspension was added to each well and the control well without sample. After incubating at 37 °C in a 5% CO2 with 95% humidity incubator for 24 h, the culture medium was removed and washed with PBS solution. Added 100 μl of MTT solution (0.5 mg·mL − 1, Sigma) to each well and incubated at 37 °C for 4 h. Following the removal of the culture medium, acidified isopropanol was added in order to dissolve the formazan crystals. The optical density of formazan was measured spectrophotometrically at 570 nm using an ELISA plate reader. This absorbancevalue is proportional to the number of viable cells. Each of thespecimens was plated in triplicate MTT assays for 24 h.
Copper affects steroidogenesis and viability of human adrenocortical carcinoma (NCI-H295R) cell line in vitro
Published in Journal of Environmental Science and Health, Part A, 2020
Jana Bilcikova, Veronika Fialkova, Hana Duranova, Eva Kovacikova, Zsolt Forgacs, Agnieszka Gren, Peter Massanyi, Norbert Lukac, Shubhadeep Roychoudhury, Zuzana Knazicka
The viability of the cells exposed to CuSO4.5H2O was evaluated by the metabolic activity (MTT) assay. [64] This colorimetric assay measures the conversion of a yellow tetrazolium salt [3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), to blue formazan particles by mitochondrial succinate dehydrogenase of intact mitochondria of living cells. Formazan was measured spectrophotometrically. Following the termination of CuSO4.5H2O exposure, the cells were stained with MTT (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 0.20 mg/mL. After incubation (37 °C, and 5% CO2 atmosphere) for 2 h, the cells and the formazan crystals were dissolved in 150 µL of acidified (0.08 M HCl) isopropanol (CentralChem, Bratislava, Slovak Republic). The absorbance was determined at a measuring wavelength of 570 nm against 620 nm as reference by a microplate reader (Anthos MultiRead 400, Austria). The data were expressed in percentage of the control group (i.e., absorbance of formazan from cells not exposed to CuSO4.5H2O).