Lasersharp

Cells cultured at low density on fibronectin-coated 35-mm glass-bottom dishes were incubated for 15 min at 37°C with 0.5 µM MitoSox (Molecular Probes, Eugene, OR) for detection of intra-mitochondrial superoxide (O₂^⋅−). Stained cells were examined with a Nikon TE 2000 microscope (images collected using a 60× objective [1.4 NA]) coupled to a Radiance 2100 dual-laser LSCM system (Bio-Rad); red fluorescence was elicited by exciting with the He-Ne laser beam (λ_ex 543 nm). Acquisition, storage, and analysis of data were performed with LaserSharp and LaserPix software from Bio-Rad or ImageJ version 1.37 (http://imagej.nih.gov/ij/). Superimposed confocal planes were analyzed by the “stack” function of the LCS-Analysis Tools which produced a xz intensity profile of the average value of the pixels within marked edges, including a single cell, as a function of each focal plane. The integrated value of the xz profile was taken as a measure of the fluorescence intensity of that individual cell relative to the selected emission channel. Correction was made for the minimal background by repeating the procedure in a cell-free field. About one hundred single cells were analyzed for each imaging analysis.

Cells cultured at low density on fibronectin-coated 35-mm glass-bottom dishes were incubated for 20 minutes at 37°C with 2 μM TMRE or 10 μM DCFH-DA (Molecular Probes, Eugene, OR) to monitor mtΔΨ and ROS respectively. Stained cells were washed with PBS and examined by a Nikon TE 2000 microscope (images collected using a 60X objective [1.4 NA]) coupled to a Radiance 2100 dual-laser (4-line Argon-Krypton, single-line Helium-Neon) confocal laser scanning microscopy system (Biorad). Acquisition, storage, and analysis of data were performed with LaserSharp and LaserPix software from Biorad or ImageJ1.48u (Wayne Rasband, NIH, USA, http://imagej.nih.gov/ij).