Antiproliferative activity and overall cytotoxicity of compound 2 on cancer and normal cells lines (breast cancer cell line MDA-MB231 and non-tumorigenic breast epithelial cells (MCF-10a) were determined in vitro using colorimetric MTT (Sigma-Aldrich (Schnelldorf, Germany)) solution (0.5 mg/mL in PBS) assay according to a protocol adapted from [44 (link)]. Cells were seeded in 96-well culture plate (1200 cells/well 1 day before incubation and were treated for 72 h in culture medium at 37 °C with different concentrations of compound 2 ranging from 1 nM to 10 µM, with semilog concentration increasing of the compound. The yellow MTT product was converted to a purple formazan derivative through mitochondrial enzymatic reduction. At the end of the incubation of 4 h, the plate was centrifugated at 1200 rpm for 5 min and the MTT solution was decanted. Then, blue formazan was solubilized with DMSO and absorbance at 570 nm, which is directly proportional to the number of metabolic active cells, is determined. Experiments were carried out in sextuplicates.
Colorimetric mtt
Manufactured by Merck Group
Sourced in Germany
Colorimetric MTT is a lab equipment product that measures cell viability and proliferation. It utilizes a tetrazolium dye-based assay to quantify metabolic activity in cells.
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2 protocols using colorimetric mtt
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Antiproliferative Effects of Compound 2
2
Colorimetric MTT Assay for Cell Viability
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The colorimetric MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-Diphenyl tetrazolium bromide) (Sigma) method was used to measure cell viability. Living cells metabolize MTT salts via mitochondrial enzyme activity. In 96-well culture plates, human neutrophils (2 × 10 5 cells/well) and different concentrations of quercetin were co-incubated (1-4 h at 37°C). The medium was replaced with MTT (0.5 mg/mL) and incubated for 4h at 37 °C. After adding DMSO to dissolve the formazan crystals, optical density (OD) was measured using a spectrophotometer (560 nm). Neutrophils incubated only with RPMI-1640 (Sigma) and 50 μM H 2 O 2 (Stocco et al., 2012) (link) were used as negative and positive controls (100% viable) for cell death, respectively.
The viability of A549 cells, treated or not with NETs, was also evaluated using the MTT assay. For this purpose, the cells (5 × 10 4 /well) were incubated with quercetin (24 h), NETs (24 h), or NETs (1 h) previous to quercetin (24 h) at 37°C.
The viability of A549 cells, treated or not with NETs, was also evaluated using the MTT assay. For this purpose, the cells (5 × 10 4 /well) were incubated with quercetin (24 h), NETs (24 h), or NETs (1 h) previous to quercetin (24 h) at 37°C.
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