Measurement of intracellular reactive oxygen species

The induction of oxidative stress was measured by using both CellRox Green (Invitrogen) and CM-H₂DCFDA (Invitrogen) in separate experiments. After 24 h of treatment and followed by two washes with 1X PBS, CellRox Green was diluted to 10 μM in media without FBS, added to the cells and incubated at 37 °C for 30 mins. Two washes with 1X PBS were done before measuring fluorescence by SpectraMax M5/M5e, using excitation and emission pair of 485/520 nm. Again, media only and media with NPs were assessed to ensure interference-free measurements. For CM-H₂DCFDA, after 24 h of cell seeding, the probe was diluted to 10 μM in RPMI media without FBS and added to the cells for 40 mins at 37 °C. After the incubation cells were washed twice with PBS and nanoparticle treatment was applied. Cell imaging was done at 24 and 72 h using InCell analyzer 6000 (GE Healthcare LifeSciences) in epifluorescence mode. Four different fields were acquired for each well. CM-H₂DCFDA fluorescence (488/510 nm excitation/emission) was acquired at a laser power of 100% and exposure of 400 ms. Images were processed using FIJI software.