Hoechst 33342 dye

A549 and CaCo-2 cells were seeded at a density of 8x10⁴ cells/well on 4-chamber polystyrene vessel tissue culture-treated glass slides (BD Falcon, Italy). 24 h after seeding, cells were exposed to 200 μg/ml of 30 or 80 nm Rubipy-SiO₂ NPs for different exposure times at 37°C either in complete cell culture medium containing 10% serum or in serum-free medium. Following exposure, cells were washed 3 times in PBS to remove unbound particles, fixed with 4% (v/v) paraformaldehyde in PBS and permeabilised with 0.1% (v/v) Triton X-100 in PBS (Sigma-Aldrich, Italy). To delimit cell boundaries and discriminate between NPs present inside or outside of the cell actin filaments were stained for 40 min at room temperature with AlexaFluor 488-conjugated Phalloidin (Invitrogen, Italy) diluted 1:100 in PBS. The nuclei were counterstained with Hoechst 33342 dye (Dako, Italy), diluted 1:2000 in PBS. After staining, the cells were washed in PBS and mounted for microscopy. Images were acquired with an Axiovert 200 M inverted microscope equipped with ApoTome slide module and Axiovision 4.8 software (Carl Zeiss; Jena, Germany), using a 40×/1.0 objective lens.

CaCo-2 cells were seeded at a density of 10⁵ cells/well in 4-chamber slides (Falcon), grown for 24 h and left untreated or incubated with chlorpromazine 50 µM, dynasore 80 µM, methyl-beta-cyclodextrin 5 mM, nystatin 40 µg/ml, genistein 200 µM, or EIPA 75 µM for 1 h at 37 °C. To investigate the energy dependence of NP uptake, CaCo-2 cells were exposed to 200 μg/ml of 30 and 80 nm-sized fluorescent Rubipy-SiO₂ NPs for 3 h at 37 or 4 °C in complete cell culture medium.
Following exposure, cells were washed 3 times in PBS, fixed with 4% (v/v) paraformaldehyde in PBS and permeabilised with 0.1% (v/v) Triton X-100 in PBS (Sigma-Aldrich, Italy) before staining with AlexaFluor 488-conjugated Phalloidin (Thermo Fisher Scientific, Italy), diluted 1:100 for 40 min at RT. The nuclei were counterstained with the Hoechst 33342 dye (Dako, Italy). After staining, the cells were washed in PBS and mounted for microscopy. Images were acquired with an Axiovert 200 M inverted microscope equipped with a ApoTome slide module and Axiovision 4.8 software (Carl Zeiss; Jena, Germany), using a 40×/1.0 objective lens.