Prenatal Diagnosis and Early Treatment of Immunodeficiencies in Man
Gérard Chaouat in The Immunology of the Fetus, 2020
The nitroblue of tetrazolium (NBT) test is very useful in the screening of patients for abnormalities in phagocytic oxidative metabolism. A presumptive diagnosis of CGD can be made when phagocytes fail to reduce NBT during stimulation. The carrier state can also be detected.31 Furthermore, it has been reported that normal human granulocytes and monocytes from 16- to 19-week-old fetuses reduce NBT and generate superoxide anion following phorbol myristate acetate stimulation.5 Therefore, prenatal diagnosis of CGD can be performed on blood samples obtained at 18 weeks postfecundation.5,32, 33 In addition to the above-described tests, a chemiluminescence assay evaluating the kinetics and regulation of oxidative metabolic response to membrane stimulation in phagocytic cells ensures diagnosis of CGD.34 Prenatal diagnosis of CGD by DNA analysis has been recently reported (deletion in CGD gene, in a family where the disease had been previously identified).35
Lab-on-a-Chip-Based Devices for Rapid and Accurate Measurement of Nanomaterial Toxicity
Suresh C. Pillai, Yvonne Lang in Toxicity of Nanomaterials, 2019
Cytotoxicity assessment in cells also can be evaluated using the Microculture Tetrazolium Assay (MTA). After treating with MTA, live cells metabolize it into a purple-coloured product which can be dissolved in dimethyl sulfoxide (DMSO) and detergent for spectrometer measurement. However, there are fundamental limitations on this assay: First, not all cell lines metabolize MTA efficiently. Second, the protocol is time-consuming (2–4 hours incubation period). Third, the usage of DMSO to solubilize the end product is extremely toxic. Thus, this assay can subject lab personnel to health risks. To solve this problem, XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) is used instead of MTA. XTT, when metabolized, can produce a water-soluble purple product. Therefore, it eliminates the need of extra step to solubilize the end product with DMSO.
Anti-Aging Drug Discovery in Experimental Gerontological Studies
Shamim I. Ahmad in Aging: Exploring a Complex Phenomenon, 2017
In our experiments, we repeatedly determined the proportion of dead cells in the same “stationary aged” culture (not subcultured for 2–3 weeks) by directly examining the cells under a microscope and by taking digital images of the culture and taking cell counts on a computer display. In both cases, the cells were examined either “as is” (without any special treatment) or after adding dyes/probes commonly used for differential staining of live and dead cells (in particular, trypan blue, methylene blue, neutral red, and MTT). In many cases, the dead cell ratio detected by these methods proved to differ significantly, which casts doubt on the efficiency of such an approach to cell viability assessment in cytogerontological research. It should also be noted that some popular dyes have a number of side effects, which researchers often fail to mention. In particular, this concerns tetrazolium salts (MTT, XTT, etc.), which are inexpensive and can be used in experiments with cells of different origin, from bacteria to mammalian cells. However, some specialists consider that these probes are not optimal for assessing cell viability, even though they allow correct estimation of metabolic activity (Berridge et al. 2005). In particular, cell metabolic activity may change due to a variety of factors, even when the number of live cells in the population remains unchanged (Keepers et al. 1991).
Fe3O4 Nanopowders: Genomic and Apoptotic Evaluations on A549 Lung Adenocarcinoma Cell Line
Published in Nutrition and Cancer, 2020
Ayse Kaplan, Hatice Mehtap Kutlu, Gulsen Akalin Ciftci
The cytotoxic effects of iron oxide nanopowders were examined in A549 and L929 cells by MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide) assay (Fig. 1). The one of the most used assays to measure metabolic activity is based on tetrazolium reduction. The most common used of these analyzes is MTT assay (14). MTT assay, yellow soluble tetrazolium salt is based on the uptake of insoluble blue purple formazan by mitochondrial succinate dehydrogenase (15). The cisplatin and iron oxide nanopowders concentrations (1–1000 µM) were tested for 24, 48 and 72 h on A549 cells. The concentration interval and time dependent reduction was observed compared to control cells for both agents (Fig. 1a and b). The iron oxide nanopowders were not effective as cisplatin. However, the iron oxide nanopowders did not cause toxic effects on L929 cells at administered doses for 24, 48 and 72 h (Fig. 1c). The IC50 dose of cisplatin and IC30 dose of iron oxide nanopowders were statistically calculated using Microsoft Excel 2010 and 11.5 SPSS program (Table 1). We performed to use IC30 value of iron oxide nanopowders due to the nontoxic effects at low doses on L929 cells and due to be most effective dose at low doses as time and dose dependent in A549 cells.
Radiosensitizing Effect of Curcumin on Human Bladder Cancer Cell Lines: Impact on DNA Repair Mechanisms
Published in Nutrition and Cancer, 2022
Joyce Azzi, Anthony Waked, Jolie Bou-Gharios, Joelle Al Choboq, Fady Geara, Larry Bodgi, Mira Maalouf
Cell proliferation of bladder cancer cell lines was determined using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (Sigma-Aldrich; cat# M5655) assay according to the manufacturer’s instructions (Promega Corp., Madison, USA). This assay measures the ability of mitochondria in a metabolically active cell to induce the conversion of a tetrazolium salt in a formazan compound. Briefly, cells were seeded in 96-well microtiter plates at a density of 1.5.103 and 1.103 cells for UM-UC5 and UM-UC6, respectively. Then, plates were incubated for 24 h at 37 °C in 5% CO2 before being treated with Curc (5 and 10 µM), IR (2 Gy), and Curc in combination with IR. 24, 48, and 72 h post-treatment, the media was removed, cells were washed twice, and 20 μL of the MTT dye was added to each microwell. After 4 h, of incubation at 37 °C, 100 μL of the solubilization solution was added to solubilize the formazan crystal, and the product was left to stabilize overnight. The optical density was measured with a microplate ELISA reader (Multiskan Ex, Thermo Scientific, Massachusetts, USA) at 595 nm. The percentage of cell proliferation was presented as the absorbance of treated cells to that of the untreated cells (control).
Cytotoxicity and effects of curcumin and cinnamaldehyde hybrids on biofilms of oral pathogens
Published in Biofouling, 2021
Vanessa Rodrigues Dos Santos, Karina Sampaio Caiaffa, Warlley Campos de Oliveira, Jesse Augusto Pereira, Gabriel Flores Abuna, Carlos Roberto Polaquini, Luís Octávio Regasini, Aimée Maria Guiotti, Cristiane Duque
In the cytotoxicity tests, L929 fibroblast culture was cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics until confluence, as described by Caiaffa et al. (2017). After the formation of a monolayer for 24 h, the cells were stimulated with the CCH 7 hybrid compound and the CHX control, between 15.6 and 156 μg ml− 1, in a 96-well microplate. Cell metabolism was assessed by methyl tetrazolium (MTT) assays. Briefly, after cell exposure to the compounds for 24 h the culture medium was aspirated, and 90 μl of fresh DMEM (without FBS) and 10 μl of MTT solution (5 mg ml −1 in phosphate buffered saline) were added to each well. The plates were incubated at 37° C for 4 h. Subsequently, the culture medium with MTT solution was aspirated and the formazan crystals were solubilized with acidified isopropanol solution (0.04 N HCl). Cell viability was evaluated by spectrophotometry at a wavelength of 570 nm. The mean values were calculated for the groups and converted into a percentage of cell metabolism in relation to the negative control (DMEM), defined as having 100% cell metabolism.
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