Lab tek 2 cc2 chamber slide system

Fe-PEI NPs were allowed to aggregate for 24 h in DMEM in the presence of the fluorescent molecule DiI (1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate Molecular Probes D-282, 30 µg/mL) either on ultra-low adherence (ULA) plates (that do not allow the adhesion of aggregates) or onto cell culture-treated chamber slides (in adherent conditions). Then, the aggregates were washed to remove not-entrapped fluorescent molecules and further used for the interaction with cells. To this aim, MSC suspension was seeded (at a density of 10,000 cells/cm²) into cell culture chamber slides (Lab-Tek II CC2 chamber slide system, Nunc, Rochester, NY, USA) either directly onto adhered Fe-PEI aggregates or in the presence of the suspension of Fe-PEI aggregates pre-formed in ULA conditions. The incorporation of DiI into MSCs was evaluated after 24 h. Briefly, cells were fixed with 4% PFA for 10 min at room temperature, followed by staining with Oregon Green-labelled WGA conjugate (wheat germ agglutinin, Molecular Probes, Eugene, OR, USA, 1/200 dilution) and Hoechst 33,258, for 10 min at room temperature. Washed slides were mounted with Prolong antifade reagent and examined by confocal microscopy (TCS-SP5 from Leica).