Quantifying IONP Uptake in Chondrosarcoma

Uptake and retention of IONP in SW1353 was assessed using a previous protocol^{20 (link)}. Chondrosarcoma cells were seeded in 24 well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) at a concentration of 10⁵ cells/well and treated as described above. Afterwards, cells were washed three times with PBS, detached and centrifuged at 1700 rpm for 5 min, to eliminate the nanoparticles that were not interacting directly with the cells. The supernatant was replaced with nanoparticle free fresh medium and the cells were seeded on glass coverslips at a density of 5 × 10⁴ cells/slide and incubated for an additional 24 h. After this time, the coverslips were washed with PBS and fixed with 4% polyformaldehyde for 10 min. Afterwards, the samples were incubated for 10 min with Hoechst (Invitrogen, Thermo Fisher Scientific, Waltham, USA) at a concentration of 1 μg/mL. Following staining, coverslips were washed with PBS and mounted on a slide with glycerol. Imaging was done using the epifluorescence microscope Olympus BX-51 (Olympus, Germany) taking advantage of DOX autofluorescence, while Hoechst counterstaining of the nuclei was applied.