MTS (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation, VWR) assay was run to determine toxicity of a variety of NMs at different doses (QSH and PS) for optimal NM exposure conditions. NMs were applied to murine Hepa1-6 cells for a period of 24 hours. Briefly, cells were seeded in triplicate onto wells of a clear flat-bottom 96-well plate at a density of 34,700 cells per well and left 24 hours for attachment. After 24 hours, medium was aspirated, and 100 μl of all NM solutions was applied to wells, except controls, for a period of 24 hours in 37°C and CO2 incubator. Negative controls were kept in medium to retain complete viability, and positive controls were kept in water for cell death. All NMs were diluted in DMEM supplemented with 10% FBS at various doses, ranging from 5 nM to approximately 250 nM. After 24 hours of exposure, cells were washed twice with complete growth medium and reapplied with 100 μl of DMEM with 10% FBS. Aliquots (20 μl) of MTS were added, and background absorbance was captured at 490 nm. Plates were then incubated for 2 hours, and absorbance was checked again. Sample absorption values were normalized to that of cells exposed to complete growth medium.

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