33 cm2) to give Ω cm2. In the experiments showing a time-dependent effect of SNP exposure, the TER is expressed

as% of t0 (TER value before SNP exposure). Immunofluorescence (IF) for endosomal marker proteins was performed to label endocytic marker proteins such as clathrin heavy chain (chc: BD, 610499) or caveolin-1 (cav: SantaCruz, sc-894) as well as flotillin-1 selleckchem and -2 (BD, 610821, BD, 610383). After nanoparticle exposure, cells were fixed with methanol/ethanol in a ratio of 2:1 for 15 min at room temperature. After fixation, cells were incubated with primary antibody diluted in 1% PBSA over night at 4 °C. After three washing steps with PBS, cells were incubated with secondary antibody (Alexa Fluor 488, Invitrogen, A11029) for 1 h at room temperature. Subsequently, cells were washed three times with PBS, and nuclei were stained with Hoechst 33342 (Molecular Probes) for 5 min and washed three times. Finally, cut transwell filters were mounted with Fluoromount-G™ (Southern Biotech, Birmingham), and ibidi μ-slides were mounted with ibidi mounting medium (ibidi, Martinsried). To draw comparisons Selleck Afatinib concerning uptake behaviour and quantification between H441 in conventional monoculture and H441 kept under coculture conditions, cells were incubated with fluorescently labelled NPs (Sicastar Red:

6 μg/ml, AmorSil: 300 μg/ml) and observed with a fluorescence microscope (DeltaVision, Applied Precision). To allow comparisons, the exposure time and intensity scale were adjusted for each sample to be compared. Subsequently, mean fluorescence intensity until was measured via Fiji (http://pacific.mpi-cbg.de) and depicted as relative fluorescent unit (RFU) related to the untreated control (x-fold of untreated control). To evaluate

putative transcytosis events, H441 (in coculture with ISO-HAS-1) were incubated with Sicastar Red (60 μg/ml), AmorSil (300 μg/ml) for 48 h. Subsequently, ISO-HAS-1 were checked for internalised NPs by direct observations of images taken with a fluorescence microscope (DeltaVision, Applied Precision). Due to a high autofluorescence of the polycarbonate filter, a quantification of the fluorescent signal by measuring the intensity via Fiji was not suitable. For transmission electron microscopy (TEM), H441 were seeded on fibronectin-coated Thermanox™ coverslips (Nunc #174969, Wiesbaden, Germany) and exposed to AmOrSil for 4 h and further 20 h cultivation in fresh serum-containing medium. Subsequently, cells were fixed in 2.5% glutaraldehyde in cacodylate buffer (pH 7.2) for 30 min then fixed in 1% OsO4 for 2 h and dehydrated in graded ethanol. The coverslips with cells were carried through propylene oxide as an intermedium; then, the samples were embedded in agar 100 resin (PLANO, Wetzlar, Germany) and submitted to polymerisation at 60 °C for 48 h. Ultrathin sections were cut with an ultramicrotome (Leica, Bensheim, Germany).