Thermo varioskan flash multi detection microplate reader

To evaluate the intracellular ROS production, a non-fluorescent probe 2′,7′-Dichlorofluorescin diacetate (DCFH-DA; Sigma-Aldrich) assay was performed. Briefly, 5×10³ cells were seeded per well in 96-well plates and treated with different concentrations of api-AuNPs (0, 0.8, and 1.2 mg/mL) in triplicate for 24 h. Then, the cells were washed with PBS and stained with DCFH-DA at room temperature for 30 min. Fluorescence intensity was measured using a Thermo Varioskan Flash Multi Detection microplate reader (Thermo Fisher Scientific) with an excitation at 485 nm and emission at 537 nm, respectively. Data are presented as the mean percentage of ROS production compared to untreated cells set as 100% ROS.

The effect of CMOSs on cellular viability was assessed by using a CellTiter 96® aqueous one-solution cell proliferation assay (MTS assay) (Promega, Madison, WI, USA). The HCT116 cells were seeded at a density of 5 × 10³ cells per well using 96-well plates in triplicate, then grown for confluency in 24 h. Cells were treated with different concentrations of CMOSs (1 to 40 mg/mL) or 5-Fluorouracil (5FU, 1 to 40 µg/mL) for 24 h. After treatment, the MTS solution was added to the wells, and plates were incubated for 1 h. Absorbance was detected at 490 nm using a microplate reader (Thermo Varioskan Flash Multi Detection microplate reader, Thermo Fisher Scientific). The percentage of viable cells was calculated by normalization with the untreated cells control. The IC₅₀ value was calculated using a linear regression equation.