We seeded the HaCaT cells in a 24-well plate at a concentration of 1x105 cells /well and we left them until they reached 80% of confluence [10 (link)]. We scratched cell cultures with a 200 μL sterile pipette tip [7 (link), 22 (link)] using the designed mold (see above for details) (S1 File ). We then washed away detached cells with PBS (1X). Then, we added 1 mL of hAdMSCs conditioned medium, which was obtained from cells in the 4-6th passage according to the protocol established by Qazi et al. [7 (link)]. We made horizontal reference lines on the bottom of the plate with an ultrafine tip marker to have a grid for alignment to obtain the same field for each image acquisition run. Once we made the reference lines (approximately 3000 μm of distance), the plate was placed under a phase-contrast microscope using as guide the reference marks. We analyzed selected regions of interest using a Zeiss inverted microscope on a 10X objective with NA/0.25 every 2 hours for 28 hours. We determined the scratch area, wound coverage of total area, and average and standard deviation of the scratch width with the aid of our plugin. We calculated the rate of cell migration (RM) and the percentage of wound closure according to (Eq 1 ) and (Eq 2 ), respectively [28 (link)]:
Where Wi is the average of the initial wound width, Wf is the average of the final wound width both in μm and t is the time span of the assay in hours. Additionally, At = 0 is the initial wound area, At = Δt is the wound area after n hours of the initial scratch, both in μm2.
Where Wi is the average of the initial wound width, Wf is the average of the final wound width both in μm and t is the time span of the assay in hours. Additionally, At = 0 is the initial wound area, At = Δt is the wound area after n hours of the initial scratch, both in μm2.
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