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To assess the toxicity of quercetin on mammalian cells, resident macrophages were obtained from golden hamsters by peritoneal lavage with 10 ml of a cold RPMI 1640 medium. Cells were plated (4 × 10⁶ in 200 µl) for 1 h at 37°C in the presence of 5% CO₂, and then, non-adherent cells were removed. Macrophage monolayers were treated in triplicate with quercetin (0–640 µM) for 48 h at 37°C/5% CO₂. Controls were macrophage monolayers treated with RPMI or 1% of vehicle DMSO (the major final concentration), and the positive control for reduced cellular viability (disrupted cells) was obtained by adding 1% Triton X-100. The viability of macrophages was then assessed by measuring the mitochondrial-dependent reduction of MTT [3-(4, 5-dimethyl- 2-thiazol)-2, 5-diphenyl-2H-tetrazolium bromide)] to formazan. MTT (10 µl to 10 mg/ml) was added to cell cultures and incubated at 37°C/5% CO₂ for 3 h. The medium was removed, and formazan crystals were dissolved in 180 µl of DMSO. The absorbance was read at 570 nm using a microplate spectrophotometer (µQuant, Biotek Instruments, Inc.). The 50% cytotoxic concentration (CC₅₀) was determined by logarithmic regression analysis using GraphPad Prism 6 software.

MTT assay was conducted in accordance with the previous study with slight modifications^{78 (link)}. Briefly, MDA MB-231, MCF-10A, and 4T1 cells were harvested, counted and seeded at 1 × 10⁴ cells per well in the 96-well plate for 24 hours. The following day, cells were treated with various concentrations of the sample and incubated for 48 and 72 hours at 37 °C with 5% CO₂. After that, 20 µL of 5 mg/mL MTT (Merck, USA) reagent was added to each well and incubated for 3 hours. Next, the solution was removed and 100 µL of dimethyl sulfoxide (DMSO) was added to the wells. Then, the plate was read at 575 nm by using the microtiter plate reader (µQuant, Bio-Tek Instrument). The results were analyzed as the percentage proliferation of thecells in respect to the concentration of the samples treated.