Ects of CSE on senescence-associated changes and the synthesis of ECM components. These data should reveal further information about the potential role of cigarette smoke in cellular events of AMD.Materials and Methods Isolation of human RPE cellsFor the total study, five human donor eyes were obtained from the eye bank of the Ludwig-Maximilians-University, Munich,Effects of Smoke in RPEGermany, and were processed within 4 to 16 hours after death. The donors ranged in age between 30 and 43 years. None of the donors had a history of eye disease. Methods of securing human tissue were humane, included proper consent and approval, complied with the declaration of Helsinki, and was approved by the Department of Medicine of the Ludwig-Maximilians-University, Munich. The consent statement was written. Human retinal pigment epithelial (RPE) cells were harvested following the procedure as described previously [28,29,30]. In brief, whole eyes were thoroughly cleansed in 0.9 NaCl solution, immersed in 5 polyvinylpyrrolidone iodine (Jodobac; Bode-Chemie, Hamburg, Germany), and rinsed again in NaCl solution. The anterior segment from each donor eye was removed, and the posterior poles were examined with the aid of a binocular stereomicroscope to confirm the absence of gross retinal disease. Next, the neural retinas were carefully peeled away from the RPE-choroid-sclera using fine forceps. The eyecup was rinsed with Ca2+ and Mg2+ free Hank’s balanced salt solution, and treated with 0.25 trypsin (GIBCO, Karlsruhe, Germany) for 1 hour at 37uC. The trypsin was aspirated and replaced with Dulbecco’s modified Eagles medium (DMEM, Biochrom, Berlin, Germany) supplemented with 20 fetal calf serum (FCS) (Biochrom). Using a pipette, the media was gently agitated, releasing the RPE into the media by avoiding damage to Bruch’s membrane.ultraviolet detection and resulted in 47.1 ng nicotine/ ml cigarette smoke on average. This concentration was similar to the GSK-690693 web plasma nicotine concentration of an average smoker [43.7 ng/ml+/238] [34]. After exposure to CSE, cells were kept for 72 hours under serum free conditions. For control experiments, air was bubbled through the serum-free DMEM, pH was adjusted to 7.4, and sterile filtered as described earlier. The medium was changed at the same time points.Cell viability assayCell viability was quantified based on a two-colour fluorescence assay, 15857111 in which the nuclei of GSK864 manufacturer non-viable cells appear red because of staining by the membrane-impermeable dye propidium iodide (Sigma-Aldrich), whereas the nuclei of all cells were stained with the membrane-permeable dye Hoechst 33342 (Intergen, Purchase, NY). Confluent cultures of RPE cells growing on coverslips in four well tissue culture plates were either non-stressed or exposed to CSE. For evaluation of cell viability, cells were washed in PBS and incubated with 2.0 mg/ml propidium iodide and 1.0 mg/ml Hoechst 33342 for 20 minutes at 37uC. Subsequently, cells were analyzed with a fluorescence microscope (Leica DMR, Leica Microsystems, Wetzlar, Germany). Representative areas were documented with Leica IM 1000 software (Leica Microsystems, Heerbrugg, Switzerland), with three to five documented representative fields per well. The labelled nuclei were then counted in fluorescence photomicrographs, and dead cells were expressed as a percentage of total nuclei in the field. All experiments were run in triplicate in RPE cultures from three donors and repeated three times.Human RPE cell cul.Ects of CSE on senescence-associated changes and the synthesis of ECM components. These data should reveal further information about the potential role of cigarette smoke in cellular events of AMD.Materials and Methods Isolation of human RPE cellsFor the total study, five human donor eyes were obtained from the eye bank of the Ludwig-Maximilians-University, Munich,Effects of Smoke in RPEGermany, and were processed within 4 to 16 hours after death. The donors ranged in age between 30 and 43 years. None of the donors had a history of eye disease. Methods of securing human tissue were humane, included proper consent and approval, complied with the declaration of Helsinki, and was approved by the Department of Medicine of the Ludwig-Maximilians-University, Munich. The consent statement was written. Human retinal pigment epithelial (RPE) cells were harvested following the procedure as described previously [28,29,30]. In brief, whole eyes were thoroughly cleansed in 0.9 NaCl solution, immersed in 5 polyvinylpyrrolidone iodine (Jodobac; Bode-Chemie, Hamburg, Germany), and rinsed again in NaCl solution. The anterior segment from each donor eye was removed, and the posterior poles were examined with the aid of a binocular stereomicroscope to confirm the absence of gross retinal disease. Next, the neural retinas were carefully peeled away from the RPE-choroid-sclera using fine forceps. The eyecup was rinsed with Ca2+ and Mg2+ free Hank’s balanced salt solution, and treated with 0.25 trypsin (GIBCO, Karlsruhe, Germany) for 1 hour at 37uC. The trypsin was aspirated and replaced with Dulbecco’s modified Eagles medium (DMEM, Biochrom, Berlin, Germany) supplemented with 20 fetal calf serum (FCS) (Biochrom). Using a pipette, the media was gently agitated, releasing the RPE into the media by avoiding damage to Bruch’s membrane.ultraviolet detection and resulted in 47.1 ng nicotine/ ml cigarette smoke on average. This concentration was similar to the plasma nicotine concentration of an average smoker [43.7 ng/ml+/238] [34]. After exposure to CSE, cells were kept for 72 hours under serum free conditions. For control experiments, air was bubbled through the serum-free DMEM, pH was adjusted to 7.4, and sterile filtered as described earlier. The medium was changed at the same time points.Cell viability assayCell viability was quantified based on a two-colour fluorescence assay, 15857111 in which the nuclei of non-viable cells appear red because of staining by the membrane-impermeable dye propidium iodide (Sigma-Aldrich), whereas the nuclei of all cells were stained with the membrane-permeable dye Hoechst 33342 (Intergen, Purchase, NY). Confluent cultures of RPE cells growing on coverslips in four well tissue culture plates were either non-stressed or exposed to CSE. For evaluation of cell viability, cells were washed in PBS and incubated with 2.0 mg/ml propidium iodide and 1.0 mg/ml Hoechst 33342 for 20 minutes at 37uC. Subsequently, cells were analyzed with a fluorescence microscope (Leica DMR, Leica Microsystems, Wetzlar, Germany). Representative areas were documented with Leica IM 1000 software (Leica Microsystems, Heerbrugg, Switzerland), with three to five documented representative fields per well. The labelled nuclei were then counted in fluorescence photomicrographs, and dead cells were expressed as a percentage of total nuclei in the field. All experiments were run in triplicate in RPE cultures from three donors and repeated three times.Human RPE cell cul.