After sonification for 20 minutes in distilled water, and in cell culture medium (DMEM) with or without 10 FBS.Cytotoxicity screening1.421.76105 cells per ml were seeded in 96-well plates (Corning Title Loaded From File Costar, The Netherlands) and were incubated overnight at 37 uC and 5 CO2 to allow cell attachment. For cytotoxicity screening on Global Eucaryotic Microcarrier GEMTM (Global Cell Solutions, Virginia, USA), 26105 cells per ml were seeded in 96-well plates (Corning Costar) coated with a 5 poly (2hydroxyethyl methacrylate) (poly-HEMA) solution (Sigma, Austria) to block cell attachment onto the plate. Cultures were exposed to different concentrations of 20 nm and 200 nm PPS as well as CNTs for 4 and 24 hours. After treatment, the viability of the cells was assessed by a formazan bioreduction (MTS) assay (CellTiter 96H AQueous Non-Radioactive Cell Proliferation Assay, Promega, Germany) 23727046 according to the manufacturer’s protocol. After two hours of incubation with the MTS-solution, the absorbance was measured on a SpectraMAX plus 384 (Molecular Devices, Austria) at 490 nm. Wells without cells but with the respective medium, in which the NPs were dissolved, were used as blank control. To investigate whether the NPs interfere with the assay, an interference control ( = highest concentration of each NP without cells) was included. In addition, after exposure to CNT, cells were washed three times with pre-warmed phosphate buffered saline (PBS) (PAA) prior to adding staining solution.Mode of action of PPS in conventional cell cultureAfter exposure of cells to the PPS for 4, 8, and 24 hours, the integrity of the cell membrane was determined using the CytoToxONETMHomogeneous Membrane Integrity Assay (Promega), based on the release of lactate dehydrogenase (LDH). The fluorescence was recorded with an excitation wavelength of 560 nm and an emission wavelength of 590 nm on a FLUOstar Optima (BMG Labtech, Germany). As positive control, the cells were treated with a lysis solution of equal amounts of Triton X100 and 70 ethanol for 10 minutes at room temperature. Induction of apoptosis was assessed after treatment of cells under same conditions as for the LDH measurement by using the Title Loaded From File Caspase-Glo 3/7 Assay (Promega). Measurements were read on a Lumistar (BMG Labtech). Both assays were carried out according to manufacturer’s instructions.Materials and Methods Cell cultureAll experiments were performed on the endothelial cell line EAhy 926 which was kindly provided by Dr. C.J. Edgell [35]. Cells were cultured in high glucose Dulbeco’s Modified Earls Medium (DMEM) supplemented with 10 fetal bovine serum (FBS), 15900046 2 mMF?L2 Glutamine and 1 penicillin/streptomycin (P/S) (PAA, Austria).Assays on long-term effects in conventional cell cultureCells were plated in 25 cm2 cell culture dishes in complete DMEM and were incubated at 37 uC and 5 CO2 to allow cell attachment. After 24 hours, the media were exchanged and NPs at a final concentration of 20 mg/ ml were added. Controls received no NPs. Cell numbers and cell viability were assessed at timepoints when controls reached 100 confluence to avoid bias byNanoparticlesNon-functionalized PPS `Nanosphere Size Standards’ 1 (w/v) 20 nm and 200 nm, red fluorescent PPS `Fluoro-Max Red Aqueous Fluorescent Particles’ R25 1 (w/v) 25 nm (ThermoLong-Term Effects of Nanoparticlesgrowth inhibition. In parallel, assays on the membrane integrity and apoptosis were assessed as described above and the cells were sub-cloned in a 1:10 ra.After sonification for 20 minutes in distilled water, and in cell culture medium (DMEM) with or without 10 FBS.Cytotoxicity screening1.421.76105 cells per ml were seeded in 96-well plates (Corning Costar, The Netherlands) and were incubated overnight at 37 uC and 5 CO2 to allow cell attachment. For cytotoxicity screening on Global Eucaryotic Microcarrier GEMTM (Global Cell Solutions, Virginia, USA), 26105 cells per ml were seeded in 96-well plates (Corning Costar) coated with a 5 poly (2hydroxyethyl methacrylate) (poly-HEMA) solution (Sigma, Austria) to block cell attachment onto the plate. Cultures were exposed to different concentrations of 20 nm and 200 nm PPS as well as CNTs for 4 and 24 hours. After treatment, the viability of the cells was assessed by a formazan bioreduction (MTS) assay (CellTiter 96H AQueous Non-Radioactive Cell Proliferation Assay, Promega, Germany) 23727046 according to the manufacturer’s protocol. After two hours of incubation with the MTS-solution, the absorbance was measured on a SpectraMAX plus 384 (Molecular Devices, Austria) at 490 nm. Wells without cells but with the respective medium, in which the NPs were dissolved, were used as blank control. To investigate whether the NPs interfere with the assay, an interference control ( = highest concentration of each NP without cells) was included. In addition, after exposure to CNT, cells were washed three times with pre-warmed phosphate buffered saline (PBS) (PAA) prior to adding staining solution.Mode of action of PPS in conventional cell cultureAfter exposure of cells to the PPS for 4, 8, and 24 hours, the integrity of the cell membrane was determined using the CytoToxONETMHomogeneous Membrane Integrity Assay (Promega), based on the release of lactate dehydrogenase (LDH). The fluorescence was recorded with an excitation wavelength of 560 nm and an emission wavelength of 590 nm on a FLUOstar Optima (BMG Labtech, Germany). As positive control, the cells were treated with a lysis solution of equal amounts of Triton X100 and 70 ethanol for 10 minutes at room temperature. Induction of apoptosis was assessed after treatment of cells under same conditions as for the LDH measurement by using the Caspase-Glo 3/7 Assay (Promega). Measurements were read on a Lumistar (BMG Labtech). Both assays were carried out according to manufacturer’s instructions.Materials and Methods Cell cultureAll experiments were performed on the endothelial cell line EAhy 926 which was kindly provided by Dr. C.J. Edgell [35]. Cells were cultured in high glucose Dulbeco’s Modified Earls Medium (DMEM) supplemented with 10 fetal bovine serum (FBS), 15900046 2 mMF?L2 Glutamine and 1 penicillin/streptomycin (P/S) (PAA, Austria).Assays on long-term effects in conventional cell cultureCells were plated in 25 cm2 cell culture dishes in complete DMEM and were incubated at 37 uC and 5 CO2 to allow cell attachment. After 24 hours, the media were exchanged and NPs at a final concentration of 20 mg/ ml were added. Controls received no NPs. Cell numbers and cell viability were assessed at timepoints when controls reached 100 confluence to avoid bias byNanoparticlesNon-functionalized PPS `Nanosphere Size Standards’ 1 (w/v) 20 nm and 200 nm, red fluorescent PPS `Fluoro-Max Red Aqueous Fluorescent Particles’ R25 1 (w/v) 25 nm (ThermoLong-Term Effects of Nanoparticlesgrowth inhibition. In parallel, assays on the membrane integrity and apoptosis were assessed as described above and the cells were sub-cloned in a 1:10 ra.
