Falcon 8 well chamber slides

For visualization of NP cell interaction, cells were seeded on Falcon® 8 well chamber slides (Corning Inc., Big Flats, NY, USA) with a density of 1 × 10⁴ cells/chamber and cultivated for 48 h. Subsequently, cells were incubated with the different NP formulations diluted to a concentration of 50 µg/mL with respective cell culture medium for 6 h. After incubation, cells were washed with PBS containing Ca²⁺ and Mg²⁺ (PBS⁺⁺). For staining of cell membrane, cells were incubated with wheat germ agglutinin AF®350 conjugate (125 µg/mL, Biotium Inc., Hayward, CA, USA) for 10 min and washed once again with PBS⁺⁺. Cells were fixed with paraformaldehyde (4%, m/v) for 15 min, washed with PBS⁺⁺ and covered with Vectashield® mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) for staining of cell nuclei. For visualization, an IX81 fluorescence microscope (Olympus, Hamburg, Germany) with different filter systems was used (filter 1 for DAPI and AF®350: excitation 360–370 nm, dichroic mirror 400 nm, emission 426–446 nm; filter 2 for NP autofluorescence: excitation 460–500 nm, dichroic mirror 505 nm, emission 510–560 nm). Subsequent analysis was performed with cellSens Dimension 1.17 software (Olympus, Hamburg, Germany). For serum free experiments, cell culture medium was replaced with serum free medium 24 h before NP incubation.

A sphere formation assay was performed as previously described by Zhang et al (39 (link)) with slight modification. Falcon 8-well chamber slides (product no. 354118; Corning, Inc.) were precoated with 50 µl of LDEV-free growth factor-reduced Geltrex (cat. no. A1413201; Thermo Fisher Scientific, Inc.). Cells were plated at 3,000 cells/well in the aforementioned culture medium supplemented with 1% Geltrex. One day later, the cells were treated with 0 or 100 µM of S0859 and incubated at 37°C for 6 days to form spheres. Images of spheres at a magnification of ×10 were captured using a Keyence BZ-X700 fluorescence microscope (Keyence Corporation). To quantify sphere growth, the number of spheres was counted and Feret diameters were measured using the FIJI version of ImageJ.