Tecnai 120 tem

TK6 cells were exposed to Ag ENMs (2.5 μg/cm²: Ag Citrate, Ag_SDS, Ag_Disperbyk and Ag_Tween and 1.5 μg/cm²: Ag_Chitosan and Ag_Byk) in 100 mm Petri dishes (7.8 × 10⁶ cells/Petri dish). After 24 h cells were fixed in 2.5 % glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.3) and left overnight. Next day, cells were washed with 0.1 M Sorensen phosphate buffer (pH 7.3) and post-fixed in 1 % osmium tetroxide in deionized water. Samples were dehydrated in increasing concentrations of ethanol (from 10 to 100 %, 10 min each step, centrifuged every time at 200 g for 5 min), immersed in ethanol/Epon (1:1 v/v) mixture and embedded in pure Epon (2 h at 37 °C and polymerised for 24 h at 60 °C). Sections (~80 nm) were cut using a diamond knife on an ultra-microtome (Leica EM UC6) and mounted on copper grids. From each sample of exposed cells 5 grids with cell sections (approximate 20–40 cells per section) were prepared. Before image acquisition, sections were stained using uranyl acetate and lead citrate. All images were acquired on an FEI TECNAI 120 TEM (120 kV). Under investigation only the cells without damaged membrane (not in necrosis or advanced apoptosis) were taken. For each Ag ENM we tried to access minimum 20 images of individual ENMs. Experiments were run in triplicate.

A549 cells were grown on 35 mm Petri dishes, at a density which allowed them to reach 80% confluence at the end of the exposure period (2, 24 and 48 h). 24 h after seeding, the cells were exposed to Ag ENMs 50 nm (5.3 μg/cm², 2.7 × 10¹¹ ENMs/cm²), 80 nm (5.3 μg/cm², 0.55 × 10¹¹ ENMs/cm²) and 200 nm (10.5 μg/cm², 0.2 × 10¹¹ ENMs/cm²) for 2, 24 and 48 h. After exposure, cells were fixed in 2.5% glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.3) for at least 1 h. Afterwards, cells were washed with 0.1 M Sorensen phosphate buffer (pH 7.3) and post-fixed in 1% osmium tetroxide in deionised water for 1 h. Samples were then dehydrated in increasing concentrations of ethanol (from 70% to 100%, 10–20 min each step), immersed in ethanol/Epon (1:1 v/v) mixture and embedded in pure Epon (2 h in 37°C) and polymerised for 24 h at 60°C. Sections (~80 nm) were cut using a diamond knife on an ultra-microtome Leica EM UC6 and mounted on copper grids. Before image acquisition, sections were stained using uranyl acetate and lead citrate. All images were acquired on an FEI TECNAI 120 TEM (120 kV).