Application suite v 3.8.0 software

Reactive oxygen species (ROS) generation, increased with the oxidative stress, was explored by using the cell-permeable ROS detection reagent CM-H₂DCFDA (5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester).^{29 (link)} Cultured endothelial cells seeded on six-well plates, were incubated with 10 μm CM-H2DCFDA (37 °C for 15 min). Cells were washed three times with PBS followed by cell stimulation with the same culture media containing 20% sera at the different time points studied, for 30 min. Intracellular ROS production was monitored in a fluorescence Leica DM4000B microscope (Leica Microsystems, Wetzlar, Germany). Up to 15–20 micrographs were taken from each condition with a Leica DFC310FX camera (Leica Microsystems, Wetzlar, Germany) and the Leica Application Suite (v.3.8.0) software (Leica Microsystems, Heerbrugg, Switzerland).

The fluorescence images of chromosomes after FISH were acquired using the Leica Application Suite V.3.8.0 software on a Leica DM 2500 microscope with a Leica DFC345 FX camera (Leica Microsystems, Wetzlar, Germany). All fluorescence images of chromosomes with telomeric DNA probes were acquired using the following settings: exposure time, 7.0 s; gain, ×3.2; and gamma, 1.75. All fluorescence images of chromosomes with 19p reference DNA probes were acquired with the following settings: exposure time, 7.0 s; gain, ×1; and gamma, 2.01. The FISH signal intensities of the telomeric and 19p subtelomeric probes on the images were evaluated using the Image J V.1.52n software, which enabled the signal intensity to be measured in the selected area of the image. Each signal area was manually selected with the Freehand Selection tool of Image J V.1.52n. Each pixel in the selected area of the image was automatically assigned a value from 0 to 255, depending on its grayscale value in 8-bit mode. The average fluorescence intensity of the selected area was calculated automatically by Image J V.1.52n by summing the values assigned to all pixels and dividing the resulting sum by the number of pixels.