Ez cytox

Cell proliferation was analyzed with a water-soluble tetrazolium formazan (WST)-based assay with EZ-Cytox (Dogen, Seoul, Republic of Korea), in accordance with the manufacturer’s instructions. Briefly, cells were plated into 96-well plates at a density of 5 × 10³ cells per well and incubated for 48 h. The cells were serum-starved for 4 h, pretreated with the inhibitors for 30 min, and then treated with gintonin, LPA, or EGF for 24 h [29 (link)]. After replacing the culture medium with fresh serum-free medium without phenol red, the cells were then treated with the EZ-Cytox solution for 2 h. Absorbance was measured at 450 nm using a Spectra Max 190 plate reader (Molecular Devices, Sunnyvale, CA, USA).

The cell viability was tested using the EZ-Cytox (Daeil Lab, Seoul, Korea) reagent according to the manufacturer’s recommendations. Briefly, HeLa cells (5 × 10³ cells/well) were cultured in 96-well plates and pretreated with human recombinant IFN-γ for 24 h. After following an overnight incubation, each of the cells was treated with serially diluted flavonoids (4–6) for 6 h. EZ-Cytox (10 µL) was added to each of the wells of the plate and incubated for 1 h. The absorbance was determined at 450 nm with SpectraMAX-190 ELISA reader (Molecular Devices, Sunnyvale, CA). The cell viability was normalised to the control group.

The in vitro cytotoxicity of NH₂‐MIL‐101(Fe) was assessed in MCF‐7 and L929 cells by using a water‐soluble tetrazolium salt‐based cell viability assay (EZ‐Cytox; Daeil Lab Service, Korea).⁴¹ Briefly, MCF‐7 or L929 cells were seeded into a 96‐well cell plate at a density of 1.5 × 10⁴ cells/well in DMEM or RPMI containing 10% FBS and 1% penicillin–streptomycin solution, respectively, which was incubated at 37°C with 5% CO₂ for 1 day. Next, the NH₂‐MIL‐101(Fe) suspension prepared in the culture medium at various concentrations (10, 25, 50, 100, 250, 500, 1000, and 1500 μg/ml) was added to each well. After another 24 h of incubation, the culture medium was removed and replaced with an equal volume of fresh medium, to which 10 μl of EZ‐Cytox reagent was added and incubated for 2 h. The plate was then assessed by measuring the absorbance at wavelengths of 450 and 600 nm using a microplate reader (SpectraMax 190 Microplate Reader; Molecular Devices, San Jose, CA). Cell viability was calculated using the following equation: Cell viability (%) = (absorbance at 450 nm of the treated well – absorbance at 600 nm of the treated well)/(absorbance at 450 nm of the untreated control well – absorbance at 600 nm of the untreated control well) × 100.⁴²