TureThe human RPE cell suspension was added to a 50 ml flask (Falcon, Wiesbaden, Germany) containing 20 ml of DMEM supplemented with 20 FCS and maintained at 37uC and 5 CO2. Epithelial origin was confirmed by immunohistochemical staining for cytokeratin using a pan-cytokeratin antibody (SigmaAldrich, Deisenhofen, Germany) [31]. RPE cells were characterized by positive immunostaining with RPE65-antibody, a RPEspecific marker (anti-RPE65, Abcam, Cambridge, UK), and quantified by flow cytometry showing that nearly 100 of cells were RPE65 positive in each cell culture. The cells were tested and found free of contaminating macrophages (anti-CD11, SigmaAldrich) and endothelial cells (anti-von Willbrand factor, SigmaAldrich). The expression of zonula occludens-1 (ZO-1; Molecular Probes, Darmstadt, Germany) was used as a marker of RPE tight junctions. After reaching confluence, primary RPE cells were subcultured and maintained in DMEM supplemented with 10 FCS at 37uC and in 5 CO2. Confluent primary RPE cells of GMX1778 web passage 3 to 5 were exposed to cigarette smoke extract (CSE) in a concentration from 2, 4, 8 and 12 for 24 hours. To generate aqueous CSE, the smoke of commercially available filter cigarettes (Marlboro, Philip Morris GmbH, Berlin, Filgotinib web Germany; nicotine: 0.8 mg; tar: 10 mg) was bubbled through 25 ml prewarmed (37uC) serum-free DMEM as described in Bernhard et al. [26]. The cigarettes were syringe-smoked in a similar apparatus as described by Carp and Janoff [32] at a rate of 35 ml/2 sec followed by a pause of 28 sec. This rate of smoking should simulate the smoking habits of an average smoker [33]. The resulting suspension was adjusted to pH 7.4 with concentrated NaOH and then filtered through a 0.22-mM-pore filter (BD biosciences filter Heidelberg, Germany) to remove bacteria and large particles. This solution, considered to be 100 CSE, was applied to RPE cultures within 30 min of preparation. CSE concentrations in the current study ranged from 2 to 12 . CSE preparation was standardized by measuring the absorbance (OD, 0.8660.05) at a wavelength of 320 nm. The pattern of absorbance (spectrogram) observed at l320 showed insignificant variation between different preparations of CSE. The nicotine in the CSE was determined by high-performance liquid chromatography withAssessment of lipid peroxidationOxidative stress can be assessed by markers of lipid peroxidation. A sensitive and specific assay for lipid peroxidation is based on metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid (PNA), a natural 18-carbon fatty acid with four conjugated double bonds, into membrane phospholipids of cells [35,36]. Oxidation of PNA results in disruption of the conjugated double bond system that cannot be re-synthesized in mammalian cells. Therefore, lipid peroxidation was estimated by measuring loss of PNA fluorescence. 24786787 Briefly, treated cells were incubated with 10 mM PNA (Molecular Probes, Invitrogen, UK) at 37uC for 30 minutes in the dark. The media was then removed and cells washed three times with phosphate-buffered saline (PBS). Afterwards, cells were scraped into 2 ml PBS using a rubber policeman. The suspension was then added to a fluorescence cuvette and measured at 312-nm excitation and 455-nm emission. A blank (unlabelled cells) was measured and subtracted from all readings. This method has been validated by treating the RPE cultures with different concentrations of hydrogen peroxide. A dose-dependent lo.TureThe human RPE cell suspension was added to a 50 ml flask (Falcon, Wiesbaden, Germany) containing 20 ml of DMEM supplemented with 20 FCS and maintained at 37uC and 5 CO2. Epithelial origin was confirmed by immunohistochemical staining for cytokeratin using a pan-cytokeratin antibody (SigmaAldrich, Deisenhofen, Germany) [31]. RPE cells were characterized by positive immunostaining with RPE65-antibody, a RPEspecific marker (anti-RPE65, Abcam, Cambridge, UK), and quantified by flow cytometry showing that nearly 100 of cells were RPE65 positive in each cell culture. The cells were tested and found free of contaminating macrophages (anti-CD11, SigmaAldrich) and endothelial cells (anti-von Willbrand factor, SigmaAldrich). The expression of zonula occludens-1 (ZO-1; Molecular Probes, Darmstadt, Germany) was used as a marker of RPE tight junctions. After reaching confluence, primary RPE cells were subcultured and maintained in DMEM supplemented with 10 FCS at 37uC and in 5 CO2. Confluent primary RPE cells of passage 3 to 5 were exposed to cigarette smoke extract (CSE) in a concentration from 2, 4, 8 and 12 for 24 hours. To generate aqueous CSE, the smoke of commercially available filter cigarettes (Marlboro, Philip Morris GmbH, Berlin, Germany; nicotine: 0.8 mg; tar: 10 mg) was bubbled through 25 ml prewarmed (37uC) serum-free DMEM as described in Bernhard et al. [26]. The cigarettes were syringe-smoked in a similar apparatus as described by Carp and Janoff [32] at a rate of 35 ml/2 sec followed by a pause of 28 sec. This rate of smoking should simulate the smoking habits of an average smoker [33]. The resulting suspension was adjusted to pH 7.4 with concentrated NaOH and then filtered through a 0.22-mM-pore filter (BD biosciences filter Heidelberg, Germany) to remove bacteria and large particles. This solution, considered to be 100 CSE, was applied to RPE cultures within 30 min of preparation. CSE concentrations in the current study ranged from 2 to 12 . CSE preparation was standardized by measuring the absorbance (OD, 0.8660.05) at a wavelength of 320 nm. The pattern of absorbance (spectrogram) observed at l320 showed insignificant variation between different preparations of CSE. The nicotine in the CSE was determined by high-performance liquid chromatography withAssessment of lipid peroxidationOxidative stress can be assessed by markers of lipid peroxidation. A sensitive and specific assay for lipid peroxidation is based on metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid (PNA), a natural 18-carbon fatty acid with four conjugated double bonds, into membrane phospholipids of cells [35,36]. Oxidation of PNA results in disruption of the conjugated double bond system that cannot be re-synthesized in mammalian cells. Therefore, lipid peroxidation was estimated by measuring loss of PNA fluorescence. 24786787 Briefly, treated cells were incubated with 10 mM PNA (Molecular Probes, Invitrogen, UK) at 37uC for 30 minutes in the dark. The media was then removed and cells washed three times with phosphate-buffered saline (PBS). Afterwards, cells were scraped into 2 ml PBS using a rubber policeman. The suspension was then added to a fluorescence cuvette and measured at 312-nm excitation and 455-nm emission. A blank (unlabelled cells) was measured and subtracted from all readings. This method has been validated by treating the RPE cultures with different concentrations of hydrogen peroxide. A dose-dependent lo.