Images plus v 2

For the evaluation of collective cell migration, an in vitro wound-healing assay was performed according to a previously described method [34 (link),35 (link)]. H1975 cells were seeded in 24-well plates at a density of approximately 5.5 × 10⁴ cells/well and incubated for 21 h in complete cell culture medium. E3330 was then added to the well at a concentration of 10 μM and incubated for 3 h. Afterwards, the cell culture medium was removed, and a scratch was performed using a 200 μL sterile pipette tip on the cell monolayer. Cells were then washed twice with warm PBS, in order to remove cellular debris, and were left to migrate in cell culture medium containing 2% FBS in the presence of cisplatin (1 μM) and E3330 (10 μM) for further 20 h. Wound closure was evaluated using a Motic AE2000 Inverted Phase Contrast Microscope (Motic, Barcelona, Spain) and pictures of the same areas were captured using a magnification of 40× with a camera Moticam 2500 (Motic, Barcelona, Spain). The scratch width was measured with Motic Images plus v2.0 software (Motic, Barcelona, Spain) at 0, 8, and 20 h after the scratch was performed. The percentage of cell migration was measured in relation to the initial distance between the wound edges. At each time-point, two pictures of the scratch were taken for each condition. Three independent experiments were performed.

Hind paw fragments collected were fixed in Bouin’s solution, decalcified for 45 days in 5% EDTA, processed for paraffin embedding, sectioned (4 μm) and stained with hematoxylin-eosin (H&E) or sirius red by the picrosirius technique to assess the presence of collagen. Cellular profile was scored by counting the different cell types (macrophages, vacuolated macrophages, fibroblasts and lymphocytes) in H&E-stained paw sections, analyzed with a photomicroscope (Olympus, Miami, FL, USA) at a final magnification of 200x. Five images from each mouse were captured using Motic Images Plus v.2.0 (Motic China Group Co. Ltd., Xiamen, China). These images were divided into four quadrants, two of which were randomly selected for cell quantification with ImageJ 1.45 s software (NIH, USA—2011). Collagen quantification of the lesion site was determined in sirius red-stained paw sections under polarized light using a photomicroscope (Nikon Eclipse 80i) with a camera (Nikon DSFi1C) coupled to a computer using Nis Element software (Shinjuku, Japan), at a final magnification of 200x. Four images of four sections from each mouse were considered for the study and analyzed by Image Pro Plus (version 4.5). The results were expressed as percentage of area with the presence of collagen compared to the total measured area.