Axio observer inverted microscope

Epithelial cells after the first passage were seeded on collagen-coated 24-well plates in M199 with P/S and 10% NCS. After reaching full confluence, cells were starved for 4 h in serum-free medium. After this time, using 10-µl pipette tips, a 500-µm wide gap was manually created in the middle of each well. Wells were washed with pre-warmed PBS and fresh M199 medium with 0.1% BSA, and single chemokines (1 ng/ml) were added into each well. Each treatment was performed in duplicate (n = 5). Controls assigned as non-treated cells were also performed. An Axio Observer inverted microscope with ZEN 2.3 software was used to monitor the experiment (Zeiss, Germany). Pictures of whole gaps were taken every 2 hours in ‘tiles mode’. For exact analysis, pictures at 0, 6, 10, 18, and 24 h of the experiment were used. The parameter that was used for statistical analysis was calculated as the percent of gap closure in the subsequent hours of the experiment. The results were analysed in comparison to the control group without additional stimuli. An additional control group with proliferation stimulator (NCS) was included to distinguish the proliferative effect from the migratory response in the designed assay.

SVF cells were pre-incubated with RGD (100 μg) in suspension or with mitomycin C (100 μg mL⁻¹, Sigma, Portugal) as a positive control of apoptosis, prior seeding at a density of 1.5 × 10⁶ cells/well in a six-well plate. After 24 h, cells were collected for western blot analysis or stained with Picogreen (1:1000, Invitrogen, USA) for 20 min at RT. Nuclei images were taken using an Axio Observer inverted Microscope with the ZEN Blue 3.2 software.