Supporting information for “Analysis of Cellular Responses of Macrophages to Zinc Ions and Zinc Oxide Nanoparticles: A Combined Targeted and Proteomic

Fixation was initiated by adding an equal volume of fixative solution, previously warmed to 37°C, to the cells after treatment with zinc oxide nanoparticles for 24 hours. The fixative solution contained 5% glutaraldehyde (Electron Microscopy Sciences. Euromedex, Strasbourg, France) in a 0.1 M sodium cacodylate buffer (both Merck, Darmstadt, Germany) (305 mOsm. pH 7.3). After 10 min the mixture was centrifuged, the supernatant was discarded, and the pellet resuspended in the fixative solution containing 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 45 min at room temperature. The cells were then washed in 0.1 M sodium cacodylate buffer, postfixed for 1 h at 4°C with 1% osmium tetroxide (Merck) in the same buffer and stained for 1 h at 4°C in 4% uranyl acetate. After further washing in distiled water. the cells were dehydrated in graded (50, 70, 80, 95, and 100%) ethanol solutions, incubated for 1 h in Epon (Electron Microscopy Sciences):absolute alcohol (1:1. vol/vol), then overnight in Epon and embedded in Epon. Ultrathin sections, stained with lead citrate (Leica, Bron, France) and uranyl acetate (Merck), were examined under a Philips CM 120 BioTwin electron microscope (120 kV).