Hoechst33342 dye blue

For confocal imaging analysis, we followed the protocol published before [17 (link)]. Briefly, 1 × 10⁵ cells were grown on glass coverslips with polylysine, incubated with mNPs for 24 h, and then washed twice with 1 mL of PBS, fixed for 20 min in 3.7% paraformaldehyde in PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were treated in a blocking buffer (3% BSA and 0.1% Triton X-100 in PBS) for 1h at rtand stained with phalloidin for 1 h. Hoechst33342 dye (Blue) (10 µg/mL) (Invitrogen, Waltham, MA, USA) was used for nuclei staining for 15 min incubation at rt. An anti-fade mounting medium (Biomeda, Foster City, CA, USA) was used for observing the labeled cells under a Bio-Rad MRC 1024 ES Confocal Laser Scanning Microscope (Bio-Rad, Hercules, CA, USA) equipped with a 15 mW Krypton/Argon laser source for fluorescence measurements (Figure 1). Cells were examined with a Nikon Plan Apo X60oil immersion objective using an excitation wavelength appropriate for Alexa 488 (495 nm). A series of optical sections (XY: 512 × 512 pixels) were then taken through the depth of the cells with a thickness of 1 µm at intervals of 0.8 µm (Z step). A single composite image was obtained by the superimposition of twenty optical sections for each sample [17 (link)].

The 5 × 10⁴ OKG4 cells/well in DermaLife medium containing 60 μM Ca²⁺ were seeded to 8-well Permanox slides (Nunc) and cultured for 24h. The cells were subsequently cultivated in 60 μM or 1.4 mM Ca²⁺ for further 24 h and treated with the respective cell culture medium containing ICC-coupled CMS 10-E-15-350 at a final ICC concentration of 2 μg/ml for further 24 h. The cell nuclei were stained with Hoechst 33342 dye (blue, Invitrogen) and cell membranes were stained with Wheat Germ Agglutinin (WGA) Alexa Fluor 488-conjugated (Invitrogen), which binds to N-acetylglucosamine and N-acetylneuraminic acid moieties on the cell surface, for 30 min. Subsequently, the cells were rinsed with PBS to remove the excess of dyes. The internalization was observed using the LSM700MAT (Zeiss) and analyzed by the ZEN software (Zeiss). Each analysis was performed in triplicate.