Remote capture dc

The cell migration was assessed using a wound assay [25 (link)]. Cells (2 × 10⁵ cells/well) were seeded into each well of a 6-well plate and incubated with complete medium at 37 °C and 5% CO₂. After 24 h of incubation, the cells were scrapped horizontally and vertically with a sterilized P10 pipette tip, subjected to different treatments in medium with 0.5% FBS and two views on the cross were photographed on each well at 0, 12, and 24 h, using a Zeiss Axiovert 40 CFL inverted microscope (Carl Zeiss, Milan, Italy) 10 × objective. The microscope was equipped with a 12.1-megapixel CCD digital video camera (Canon, PowerShot G9, Italy) with a digital image software (Remote Capture DC, Canon). Quantitative analysis of the scratch assay was performed by measuring the gap area using the free image-processing software ImageJ version 1.47.

Cell viability was evaluated as mitochondrial metabolic activity [57 (link)] using the MTT assay as previously reported [10 (link)]. Briefly, the cells were plated onto 96-well plates (1 × 10⁴ cells/well), in DMEM with 10% FBS. After 24 h seeding, the cells were treated with different concentrations of AFPE or DMEM containing 0.2% PBS. After 24 h, 10 μL of the MTT solution (5 mg/mL) was added to each well in the dark, and the plates were incubated for 3 h at 37 °C under 5% CO₂ atmosphere. Following medium aspiration, double washing with 100 µl PBS, and solubilization of formazan crystals with 250 µL DMSO, the absorbance was measured at a wavelength of 570 nm using an ELISA plate reader (BioRad, Milano, Italy). The cell viability was expressed as a percentage relative to the untreated cells, cultured in a serum-free medium, set as 100%.
To evaluate cell morphology, the cells were seeded subconfluently onto a 24-multiwell, treated with different AFPE concentrations or in DMEM containing 0.2% PBS, and then observed for 24 h by a phase-contrast Zeiss Axiovert 40 CFL inverted microscope (Carl Zeiss, Milan, Italy) using a LD A-Plan 10×/0.50 P h 2 objective and equipped with a 12.1-megapixel CCD digital capture camera (Canon, PowerShot G9, Italy). Images were acquired using digital image software (Remote Capture DC, Canon).