Application suite integrated software las x

Cells cultured at different stage of differentiation (0–7–14–21 days) on fibronectin-coated 35-mm glass-bottom dishes (Eppendorf, Hamburg, Germany) were incubated for 20 min at 37 °C with either 2 μM TMRE (Tetramethylrhodamine ethyl ester perchlorate) to monitor mitochondrial membrane potential (mtΔΨ) or with 10 μM DCF-DA (2,7-dichlorofluorescin diacetate) or 5 μM MitoSox to evaluate intracellular peroxide and mitochondrial O₂^•− respectively. In particular, MitoSox is a probe chemically engineered to target the mitochondrial matrix by adding a liposoluble cation (TPP⁺) moiety to hydroethidine which, once oxidized, enhances fluorescence following intercalation in double stranded DNA. Conversely DCF, derived from DCF-DA de-acetylation by cellular esterases, is a redox-sensitive probe, trapped within the cell in the cytosol as well as into mitochondria. Fluorescent dyes were purchased from Molecular Probes (Eugene, OR, USA). Stained cells were washed with PBS and examined by a Leica TCS SP8 confocal laser scanning microscopy system as described elsewhere [35 (link)] (images collected using a 60X objective [1.4 NA]). Acquisition, storage, and data analysis were performed using the Leica Application Suite integrated software (LAS-X, Leica Microsystems, Wetzlar, Germany) and further analyzed by the ImageJ software (Wayne Rasband, NIH, USA, http://imagej.nih.gov/ij).

Cells cultured at low density on fibronectin-coated 35-mm glass-bottom dishes (Eppendorf, Hamburg, Germany) were incubated for 20 min at 37 °C with (a) 2 μM TMRE (Tetramethylrhodamine ethyl ester perchlorate) or 0.5 μM MitoTracker Red to monitor mtΔΨ, (b) 10 μM DCF (dichlorofluorescein) to evaluate ROS production. Fluorescent dyes were purchased from Molecular Probes (Eugene, OR, USA). Stained cells were washed with PBS and examined using a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). Acquisition, storage, and data analysis were performed using the Leica Application Suite integrated software (LAS-X, Leica Microsystems, Wetzlar, Germany) and the ImageJ software (Available online: https://imagej.nih.gov/ij/download.html (accessed on 10 April 2020)).