Ss of fluorescence could be observed (data not shown). All experiments were run in triplicate in RPE cultures from three donors and repeated three times.Senescence-associated ?DMXAA galactosidase activityThe proportion of RPE cells positive for the senescenceassociated ?galactosidase (SA-?Gal) activity was determined as described by Dimri et al. [37]. Briefly, treated RPE cells were washed twice with PBS and fixed with 2 formaldehyde and 0.2 glutaraldehyde in PBS at pH 6.0 at room temperature (RT) for 4 minutes. Cells were then washed twice with PBS and incubated under light protection for 8 hours at 37uC with 1531364 fresh SA-?Gal staining solution (1 mg/ml 5-bromo-4-chloro-3-indoyl-?D-galactopyranoside (X-gal), 40 mM 23115181 citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyEffects of Smoke in RPEanide, 150 mM NaCl, 2 mM MgCl2 diluted in PBS). Cells were then examined for the development of blue color and photographed at low magnification (2006) using a light microscope. All experiments were run in triplicate in RPE cultures from three donors and repeated three times.were run in triplicate with three different RPE cultures from three donors.Analysis of fibronectin and laminin secretion into culture mediaRelease of fibronectin and laminin into culture media of RPE cells was measured using corresponding QuantiMatrixTM Human Fibronektin ELISA kits (Millipore, Billerica, MA, USA) and QuantiMatrixTM Human Laminin ELISA kits (Millipore) according to the manufacturer’s instructions. All experiments were run in triplicate with three different RPE cultures from three donors.RNA isolation and real-time PCRTotal RNA was isolated from 10 mm petri dishes by the guanidium thiocyanate-phenol-chloroform extraction method (Stratagene, Heidelberg, Germany). Structural integrity of the RNA samples was confirmed by electrophoresis in 1 Trisacetate-EDTA (TAE)-agarose gels. Yield and purity were determined photometrically. After RNA isolation, mRNA was transcribed to cDNA via reverse transcriptase. This cDNA was then used for specific real-time PCR. Quantification of human mRNA was performed with specific primers during 40 cycles with a LightCycler Instrument (LightCycler System, Roche Diagnostics, Mannheim, Germany). The primers selected were apolipoprotein J (Apo J) forward primer 59- ggacatccacttccacagc -39 and reverse primer 59- ggtcatcgtcgccttctc -39; connective tissue growth factor (CTGF) forward primer 59- ctgcaggctagagaagcagag -39 and reverse primer 59- gatgcactttttgcccttct -39; fibronectin forward primer 59-ctggccgaaaatacattgtaaa-39 and reverse primer 59ccacagtcgggtcaggag-39; and GAPDH forward primer 59-agccacatcgctcagacac-39 and reverse primer 59-gcccaatacgaccaaatcc-39. Primers and probes were found with the programme ProbeFinder Version: 2.04. The standard curve was obtained from probes of three different untreated human RPE cell cultures. To normalize differences of the amount of total RNA added to each ASA-404 reaction, GAPDH was simultaneously processed in the same sample as an internal control. The level of Apo J, CTGF and fibronectin mRNA was determined as the relative ratio (RR), which was calculated by dividing the level of Apo J, CTGF and fibronectin mRNA by the level of the GAPDH housekeeping gene in the same samples. All experiments were run in triplicate in RPE cultures from three donors and repeated three times.Statistical analysisResults for the analyses of RPE cell death, lipid peroxidation, SA-?Gal activity, real-time.Ss of fluorescence could be observed (data not shown). All experiments were run in triplicate in RPE cultures from three donors and repeated three times.Senescence-associated ?galactosidase activityThe proportion of RPE cells positive for the senescenceassociated ?galactosidase (SA-?Gal) activity was determined as described by Dimri et al. [37]. Briefly, treated RPE cells were washed twice with PBS and fixed with 2 formaldehyde and 0.2 glutaraldehyde in PBS at pH 6.0 at room temperature (RT) for 4 minutes. Cells were then washed twice with PBS and incubated under light protection for 8 hours at 37uC with 1531364 fresh SA-?Gal staining solution (1 mg/ml 5-bromo-4-chloro-3-indoyl-?D-galactopyranoside (X-gal), 40 mM 23115181 citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyEffects of Smoke in RPEanide, 150 mM NaCl, 2 mM MgCl2 diluted in PBS). Cells were then examined for the development of blue color and photographed at low magnification (2006) using a light microscope. All experiments were run in triplicate in RPE cultures from three donors and repeated three times.were run in triplicate with three different RPE cultures from three donors.Analysis of fibronectin and laminin secretion into culture mediaRelease of fibronectin and laminin into culture media of RPE cells was measured using corresponding QuantiMatrixTM Human Fibronektin ELISA kits (Millipore, Billerica, MA, USA) and QuantiMatrixTM Human Laminin ELISA kits (Millipore) according to the manufacturer’s instructions. All experiments were run in triplicate with three different RPE cultures from three donors.RNA isolation and real-time PCRTotal RNA was isolated from 10 mm petri dishes by the guanidium thiocyanate-phenol-chloroform extraction method (Stratagene, Heidelberg, Germany). Structural integrity of the RNA samples was confirmed by electrophoresis in 1 Trisacetate-EDTA (TAE)-agarose gels. Yield and purity were determined photometrically. After RNA isolation, mRNA was transcribed to cDNA via reverse transcriptase. This cDNA was then used for specific real-time PCR. Quantification of human mRNA was performed with specific primers during 40 cycles with a LightCycler Instrument (LightCycler System, Roche Diagnostics, Mannheim, Germany). The primers selected were apolipoprotein J (Apo J) forward primer 59- ggacatccacttccacagc -39 and reverse primer 59- ggtcatcgtcgccttctc -39; connective tissue growth factor (CTGF) forward primer 59- ctgcaggctagagaagcagag -39 and reverse primer 59- gatgcactttttgcccttct -39; fibronectin forward primer 59-ctggccgaaaatacattgtaaa-39 and reverse primer 59ccacagtcgggtcaggag-39; and GAPDH forward primer 59-agccacatcgctcagacac-39 and reverse primer 59-gcccaatacgaccaaatcc-39. Primers and probes were found with the programme ProbeFinder Version: 2.04. The standard curve was obtained from probes of three different untreated human RPE cell cultures. To normalize differences of the amount of total RNA added to each reaction, GAPDH was simultaneously processed in the same sample as an internal control. The level of Apo J, CTGF and fibronectin mRNA was determined as the relative ratio (RR), which was calculated by dividing the level of Apo J, CTGF and fibronectin mRNA by the level of the GAPDH housekeeping gene in the same samples. All experiments were run in triplicate in RPE cultures from three donors and repeated three times.Statistical analysisResults for the analyses of RPE cell death, lipid peroxidation, SA-?Gal activity, real-time.
