Ts2r fl fluorescence inversion microscopy

Live and dead cells were observed using a calcein-AM/PI cell viability/cytotoxicity assay kit and a fluorescence microscope, and 5 × 10⁵ CT-26 cells were seeded into 6-well plates with 2 mL DMEM complete medium per well. CT-26 cells were incubated with PBS, DMSNs, DSF@DMSNs, Zn-DMSNs and DSF@Zn-DMSNs at a concentration of 1.5 μg/mL for 24 h. After removing the culture medium and washing with PBS, live cells were stained with green calcein-AM fluorescence, and dead cells were stained with red PI fluorescence for 15 min. The staining solution was removed, and the plates were washed with PBS three times. Next, TS2R-FL fluorescence inversion microscopy (Nikon, Tokyo, Japan) was used to observe and take pictures.

To examine the cellular uptake of DMSNs and Zn-DMSNs, 5 × 10⁵ CT-26 cells were seeded into 6-well plates with 2 mL of DMEM complete medium for 24 h and then incubated with FITC-labeled nanoparticles at a concentration of 1.5 μg/mL for 2 h, 4 h and 8 h. Next, the cells were fixed with 4% paraformaldehyde (POM) and stained with DAPI. Fluorescence graphs at different time points were acquired by TS2R-FL fluorescence inversion microscopy (Nikon, Tokyo, Japan).