0.1 m sodium cacodylate

A549 cells were incubated with TiO₂ NPs (800 μg mL⁻¹, 37 °C for 1 h or 24 h and 4 °C for 10 min). Cells were processed and imaged by the Electron Microscopy Laboratory, Department of Pathology, Duke University. Thin cell sections for analysis were obtained from A549 cells fixed with 3% glutaraldehyde (#16220, Electron Microscopy Sciences, Hatfield, PA) in 0.1 M sodium cacodylate (#12300, Electron Microscopy Sciences). Samples were washed three times with 0.1 M cacodylic acid buffer and post-stained with 1% osmium tetroxide (#19190, Electron Microscopy Sciences, Hatfield, PA) in cacodylic buffer for 1 h. Cells were embedded in 1% agarose (#V2111, Promega Corporation, Madison, WI). The agarose containing the cell sample was pre-stained with 1% uranyl acetate (#22400, Electron Microscopy Sciences, Hatfield, PA) overnight at 4 °C. The samples were dehydrated using acetone. Infiltration was done using the Epon embedding kit (#14120, Electron Microscopy Sciences, Hatfield, PA). Samples were sectioned (60–70 nm) using an ultramicrotome (Ultracut E, Reichert/Leica, Wetzlar, Germany). The sections were stained with 2% uranyl acetate in 50% ethanol for 30 min and SATO's lead stain for 1 min. Samples were then imaged on a TEM (80 kV, C12, Philips, Andover, MA). 12–18 images were collected at each time point: representative images are shown.