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Advances in Regenerative Medicine and Nano-Based Biomaterials
Published in Suvardhan Kanchi, Rajasekhar Chokkareddy, Mashallah Rezakazemi, Smart Nanodevices for Point-of-Care Applications, 2022
K. Ganesh Kadiyala, P.S. Brahmanandam, Rajya Lakshmi Chavakula, Naresh Kumar Katari
The MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cytotoxicity assay is the quantitative approach recommended, as it can accurately count the metabolic activity of ≥ 950 cells. It is a colorimetric assay that detects the metabolic profile over the analysis of the number of viable cells. MTT is a water-soluble tetrazolium salt, which becomes an insoluble purple formazan product after the cleavage of the tetrazolium ring by succinate dehydrogenase, found within the cells of mitochondria. The product formazan is impermeable to the cell membranes and therefore it gathers in healthy cells. The water-insoluble product formazan can be dissolved in a solvent, like dimethyl sulfoxide. The absorbance of the resulting solution at 540 nm is directly associated with both the number of living cells and their metabolic activity [62]. The MTT assay has advantages over other assays, it enables quantitative analysis without using radioactive labeling, and it is quick and reproducible. The various biomaterials tested on different types of cells and their evaluation are represented in Table 19.3.
In Vitro: Limitations of the Main Cytotoxicity Assays
Published in Vineet Kumar, Nandita Dasgupta, Shivendu Ranjan, Nanotoxicology, 2018
Montserrat Mitjans, Daniele Rubert Nogueira-Librelotto, María Pilar Vinardell
The colorimetric MTT assay is a widely used cell viability assay based on reduction of the yellow tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a purple water-insoluble formazan in cells bearing intact mitochondria (Mosmann 1983). It has been improved and applied in numerous cytotoxicity studies, and employed to validate other methods and also to determine the toxicity of various kinds of nanomaterials. Other modified assays use tetrazolium salt viability assays, such as WST-1 (Water Soluble Tretazolium) and XTT, which reduce the viable cells to form the colored formazan molecule, or the dichlorofluorescein assay which uses a fluorescence measurement. However, the assay protocols described in the literature differ widely among them, and such discrepancies must be taken into account when comparing results reported by different laboratories (Kroll et al. 2009). Due to their optical properties, nanoparticles present in cell cultures may directly influence the readout by increasing light absorption, an effect which has already been demonstrated for sodium titanate nanoparticles (Davis et al. 2007).
Gold Nanoparticles with Organic Linkers for Applications in Biomedicine
Published in Tuan Vo-Dinh, Nanotechnology in Biology and Medicine, 2017
Olga Shimoni, Stella M. Valenzuela
The common approaches used to assess cell toxicity in in vitro systems are via cell viability assays, such as MTT assay, which assesses cell metabolic activity. The MTT assay uses the drug 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is readily taken up by cells grown in culture (Mosmann 1983). Within viable cells, the MTT is enzymatically reduced to formazan, resulting in its conversion from a yellow to purple color that is readily detected at 570 nm using a UV–Vis microplate reader. It should, however, be noted that assay systems such as MTT and others employ similar wavelengths to detect color changes by the converted drug of interest (e.g., 570 nm for formazan), which can overlap with the peak emission spectra for the Au NPs, dependent on their size, shape, and concentration (Pan et al. 2007, Kroll et al. 2012). Therefore, the imperative for inclusion of relevant controls in these assays is to exclude interference and background attributable to the Au NPs themselves.
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.
Effect of cellulose nanocrystals on chitosan/PVA/nano β-TCP composite scaffold for bone tissue engineering application
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Asif Ali, Saleheen Bano, Satish Poojary, Ananya Chaudhary, Dhruv Kumar, Yuvraj Singh Negi
MTT assay is based on spectroscopic measurement of active formazan crystals obtained on reduction of MTT dye by mitochondrial enzymes of viable cells. In other words, the method is also used to calculate the number of viable cells present in the samples. A significant increase in cell viability was recorded in the samples with nano β-TCP content .however highest cell viability was observed on incorporating 5%CNC in CS/PVA/β-TCP matrix (Figure 9). The results clearly reveal the synergistic response of CNC with nano β-TCP in osteoinduction. This can be possibly due to alteration of surface charge and creation of nano topographical cues on the scaffold. The concentration dependent change in surface texture of scaffold surface on incorporating 5% CNC favor growth and proliferation of cells, however if we increase the concentration of CNCs to 10%, there is a change in surface texture of scaffold and may result in mild agglomeration leading to decrease in cell viability.
Hydroxyapatite-collagen nanoparticles reinforced polyanhydride based injectable paste for bone substitution: effect of dopant addition in vitro
Published in Journal of Biomaterials Science, Polymer Edition, 2021
Sudip Dasgupta, Soumini Mondal, Sambit Ray, Yogendra Pratap Singh, Kanchan Maji
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a colorimetric assay that was used to assess metabolic activity of MSCs cultured onto polyanhydride samples. In brief, the samples were submerged in DMEM cell culture media and MSCs were seeded onto each sample at 1 × 107 cells per well and incubated for 3, 7, and 11 days on replacement of cell culture media with fresh DMEM after every 3 days of the culture period. After a specific cell culture period, 20 μL (0.5 mg/mL) of MTT solution was added onto each sample placed inside a 24-well plate. Another 4 h of incubation period at 37 °C. Each sample containing the MSCs and MTT solution was kept until purple-colored formazan crystals were precipitated at the bottom of 24 well plates. MTT solvent to the extent of 150 μL was added to each well to dissolve purple colored formazan crystals by shaking the plate for 15 min. The amount of produced purple colored formazan crystal and the color intensity of the resultant solution are proportional to the number of viable cells present in the sample. Then the solutions were transferred in ependrofs and centrifuged and the supernatant was poured into another 96-well plate to record the absorbance at 595 nm using a microplate reader (Bio-Tek ELx800).