Las ez application suite image analysis software

HaCaT cells were seeded at a density of about 1.5 × 10⁶ cells/well (in a 6-well plate) in complete medium at 37 °C and 5% CO₂ (v/v) and grown for 24 h to allow them to reach about 90% confluence. Before starting treatments, cells were serum-starved overnight. The monolayer of synchronized cells was gently scratched across the center of the well with a sterile pipette tip (Ø = 0.1 mm), and after scratching, cell debris was removed by washing with phosphate buffered saline (PBS, Merck KGaA, Darmstadt, Germany). Fresh medium containing 10% v/v of FBS and HA, WSCP1, WSCP2, WSCP1/HA, and WSCP2/HA, at 0.1, 1, and 10 μg/mL, were added to each well. Images of experiments conducted in triplicate were obtained from the same fields immediately after scratching (T0) and after 6, 18, and 24 h, using a Leica DMIL inverted microscope (Leica, Wetzlar, Germany) and analyzed using Leica LAS EZ Application Suite image analysis software by manually selecting the distance between the opposite edges in the wound region. Measurements were made at three different points in each image and averaged. Untreated scratched cells were considered as the control. Quantification of relative wound closure was performed according to Equation (1): $$\%\mathrm{cell}\text{-}\mathrm{free}\mathrm{area}=\frac{[\%\mathrm{cell}\text{-}\mathrm{free}\mathrm{scratched}\mathrm{area}\mathrm{at}\mathrm{T}0-\%\mathrm{cell}\text{-}\mathrm{free}\mathrm{area}\mathrm{at}\mathrm{T}6\text{-}18\text{-}24]}{\left[\%\mathrm{cell}\text{-}\mathrm{free}\mathrm{scratched}\mathrm{area}\mathrm{at}\mathrm{T}0\right]}\times 100$$

HaCaT cells were seeded in a 6-well plate at 1.5 × 10⁶ cells/well in 1.0 mL of complete medium. When the cells reached 80%, the monolayer was gently scratched across the center of the well with a sterile pipette tip (Ø = 0.1 mm). The scratch determines the onset of secondary and progressive damage to the cells. Thus, we did not change the culture medium after wounding, keeping cell debris and other factors released from the detached cells. The scratch injured cells were incubated at 37 °C and 5% CO₂ until wound closure. The WH was evaluated using phase-contrast images acquired using an inverted microscope (Leica DMi1, Wetzlar, Germany). Cell migration rate was calculated as the distance traveled by the cells, over time: just after the scratch (T0), after 6 h (T6), and after 24 h (T24). We calculated the change in a cell-free area, measuring the leading edges at the same four reference points for each well, using Leica LAS EZ Application Suite image analysis software. Three independent experiments were conducted, and four points of the wounded area were analyzed for each replicate. The percentage of the cell-free area was calculated referring to images at T0, for each sample. WH was compared between the control samples and the experimental samples by measuring the wound width.