Bacterial challenge HGEC cultures at the fourth passage were harvested and seeded at a density of 0.5 × 105 cells/well in a 6-well culture plate coated with type-I collagen or in a 35-mm collagen-coated glass bottom culture dishes (Mat-tek Corp., Ashland,

MA, USA), and maintained in 2 ml of complete medium. When they reached confluence (approximately 106 cells/well), the cells were washed twice with fresh media and were challenged with live or heat-inactivated bacteria in antibiotic-free medium at MOI:10 (107 bacteria/well) and MOI:100 (108 bacteria/well) at 37°C in 5% CO2 for 4 or 24 hours. For each experiment the final concentration H 89 order of the suspension was determined by measurement of A600 and appropriate dilutions were made to achieve the desired MOI. The click here bacterial number was confirmed by viable counting of colony forming units (cfu) on blood agar plates incubated at anaerobically at 37°C. M30 epitope detection The M30 epitope released by caspase-cleaved cytokeratin-18 was detected using a commercially available kit (CytoDEATH Fluorescein kit, Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer’s instructions. Briefly, the cells were washed three times with PBS, fixed with ice-cold pure methanol for 30 minutes at -20°C and then incubated with the M30 antibody for 60 minutes at room temperature. After three washes, the cells were observed on a confocal

microscope (Olympus Fluoview 500, Center Valley, PA, USA). Caspase-3 activity assay Caspase-3 activity was determined by FIENA (Fluorometric

however Immunosorbent Enzyme Assay) using a commercially available kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s instructions. Briefly, after Fedratinib in vivo centrifugation of the 6-well plates, the supernatant was discarded and the cells were incubated in lysis buffer for one minute on ice. After centrifugation, the cell lysate was collected, added into the anti-caspase 3 coated microplate, and incubated for 60 minutes at 37°C. After washing, the caspase substrate was added and incubated for 24 h at 37°C. The fluorescence was measured at 360/528 nm. DNA fragmentation assay Histone associated DNA fragments were detected using a commercially available kit (Cell Death Detection ELISA, Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer’s instructions. Briefly, after centrifugation of the 6-well plates, the supernatant was discarded and the cells were incubated in lysis buffer for 30 minutes at room temperature. After centrifugation, the cell lysate was collected and added into the streptavidin-coated microplate. Incubation with the monoclonal antibodies, anti-histone (biotin-labeled) and anti-DNA (peroxidase-conjugated), was followed by washing and incubation with peroxidase substrate. The absorbance was measured at 405 nm.