Falcon 4 well chamber slides

DNA cleavage of hRPE cells and U-251 glioma cells grown on Falcon 4-well chamber slides (Corning Inc, Corning, NY) was measured by TdT-mediated dUTP nick-end labeling (TUNEL; In-Situ Cell Death Detection Kit, Roche). Adherent cells from control and treated cells were processed according to the manufacturer’s protocol and cells were analyzed via confocal microscopy (LSM510, Carl Zeiss, Thornwood, NY) and fluorescence microscopy, (Keyence, Itasca, IL) with a DAPI counterstain (Vector Laboratories, Burlingame, CA).

hRPE cells were grown on Falcon 4-well chamber slides (Corning Inc.) and treated as described above. After treatment, cells were fixed with 4% paraformaldehyde for 15 minutes. Cells were then blocked and permeabilized with 5% goat serum (Invitrogen, Carlsbad, CA) and 0.3% Triton X-100 for 1 hour. Primary antibodies reactive against activated caspase 3 and caspase 4 (Abcam, Cambridge, MA) were added for overnight prior to addition of FITC-conjugated secondary antibody (Invitrogen, Carlsbad, CA) for 1 hour. Slides were mounted with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Slides were examined using a confocal microscope (LSM510, Carl Zeiss, Thornwood, NY).

Mitochondrial superoxide production was measured by using Mitosox Red Kit (Life Technologies, Carlsbad, CA) per manufacturer’s protocol. All experiments were performed in CO₂ incubator at 37°C. Cells were cultured in Falcon 4-well chamber slides (Corning Inc., Corning, NY). For confocal microscopy, Mitosox and ER tracker green (Life Technologies, Carlsbad, CA) was added at 5 μM and 500 μM, respectively, in the medium 30 minutes prior to the end of treatment. Cells were washed with PBS and briefly fixed with 4% paraformaldehyde for 1 minute prior to mounting with mounting medium (Vector Laboratories, Burlingame, CA) and imaging.