4 chamber slides

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.

IVDs were harvested from 10 rat spines for each series of cell culture experiments. The AF, NP and CEP were carefully dissected from thoracolumbar discs. IVD cells were isolated by sequential enzyme digestion with 0.4% Pronase (Calbiochem, La Jolla, CA, USA) for 1 hour (AF, CEP) or 30 minutes (NP), followed by 0.025% Collagenase P (Roche Applied Science, Mannheim, Germany) for 3 hours (AF), 30 minutes (NP) or 1 hour (CEP) in a 5% CO₂, 95% air incubator at 37 °C. Following enzyme digestion, the suspension was filtered through a 40-μm mesh (BD Falcon, Franklin Lakes, NJ, USA) for AF cells or 70-μm mesh for NP and CEP cells. The filtered cells were first washed with Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F12; Gibco, Palo Alto, CA, USA) and primary culture was begun. Isolated cells were cultured in monolayer in 4-chamber slides (BD Falcon) for immunohistochemical analysis at 1.4 × 10⁴ cells/mL or 6-well tissue culture plates (BD Falcon) at 4.0 × 10⁴ cells/mL, with 5% CO_2, 95% air in complete medium (DMEM/F12 containing 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel), 25 μg/mL ascorbic acid (Sigma-Aldrich) and 50 μg/mL gentamicin (Gibco)]. The medium was changed every third day. Primary cultured cells (in cumulative population doublings) were used in all the experiments conducted in this study.