Cytoselect 24 well cell migration assay

Cell migration was assessed using the CytoSelect^TM 24-well Cell Migration Assay (8 μm pore size, colorimetric format kit) (Cell Biolabs Inc). Mia-Paca-2 cells were seeded in the inserts (8 μm pore size) in serum-free medium containing diverse concentrations of PGG (0–10 μM) at a density of 4 × 10⁴ cells/200 μL/well. The inserts were then placed in wells containing 500 μL of medium containing 10% FBS. Cells were incubated at 37^○C for 48 h. After 48 h, the medium in the inserts was removed, and cells were carefully stained using the cell staining solution. Cells were harvested using extraction buffer, and absorbance was measured at 560 nm using a microplate reader (Sunrise; Tecan, Austria).

For migration studies, a standard assay was used to determine the number of cells that traversed a porous polycarbonate membrane in response to a chemo-attractant (higher serum concentration)^{51 (link),52 (link)} using the Cytoselect 24-well cell migration assay (Cell Biolabs). Invasion was measured using BD BioCoat Matrigel invasion chambers (BD Bioscience). In both assays, cells were transfected with AMOs, and 1.5 × 10⁵ and 2.5 × 10⁵ cells per well were seeded in an upper chamber in serum free media for the migration and invasion assay, respectively. The lower chamber was filled with media containing 10% FBS. After 24 h, cells passing through polycarbonate membrane were stained and counted according to the manufacturer’s instructions.

The CytoSelect™ 24-well cell migration assay (Cell Biolabs, San Diego, CA, USA) was applied to evaluate the PDGF-BB-mediated VSMC migration. To determine the effects of extract samples on cell migration, 1 × 10⁶ VSMCs with 50 ng/mL of PDGF-BB was placed with different concentrations of the GT extract in 100 μL of a serum-free medium on an inserted polycarbonate membrane, and then it was put into the bottom chamber that contained SMCM with 10% FBS as a chemo-attractant. After incubating for 24 h, the non-migrated cells in the upper chamber were cleared. The invaded cells were cleaned with a phosphate buffer solution (PBS—0.01 M and pH 7.2) three times, fixed with methanol, stained with 400 μL of crystal violet, and photographed under a phase-contrast microscope. The numbers of invaded cells were counted.

The in vitro migration capacity of this final cell platform towards NMU cancer cells was evaluated as previously described [23 (link),24 (link)]. Non-modified DMSC were used as positive control, and migration in the absence of NMU coculture was used as a negative control. Briefly, NMU cells were seeded in 24 well plates 24 h in advance, as was described for the coculture experiments. In this case, non-modified DMSC or the final cell-platform were seeded in Millicell culture plate inserts with 8 μm pore polycarbonate membranes (Merck Millipore, Spain). Furthermore, 3 × 10⁵ DMSC in 300 µL of serum-free DMEM were seeded in the inserts which were then placed over the wells containing the NMU cells. Migration was assessed at 24 h by the CytoSelect 24-Well Cell Migration Assay (Cell Biolabs, Bionova Cientifica, S.L., Madrid, Spain). Non-migrant cells were removed from the top of the membrane and migrant cells on the bottom of the polycarbonate membrane were stained with the cell dye solution according to the manufacturer’s instructions. Color of the stained cells was extracted and absorbance at 560 nm was quantified with the plate reader Enspire (PerkinElmer). All experiments were done in triplicate.