Fv1000 laser scanning confocal scanning microscope

MDA-MB-231 cells were seeded in a chamber slide with a density of 5 ×10⁴ cells/well in 300 μL of DMEM medium and cultured overnight. Then the cells were incubated with free DOX, Doxil^®, and DOX-loaded Rf-containing nanofromulations at the final DOX concentrations of 10 μM for free DOX and 30 μM for Doxil^® and DOX-loaded Rf-containing nanoformulations at 37 °C, respectively. After 2 h incubation, the cells were washed three times by cold PBS and fixed by 4% paraformaldehyde for 10 min. The cell nuclei were counterstained by 4’,6-diamidino-2-phenylindole (DAPI). The slides were mounted with cover slips and cells were imaged with a Nikon FV1000 laser scanning confocal scanning microscope.

The cellular uptake and intracellular trafficking of the protein-incorporated NPs were determined by fluorescence microscopy. FITC-BSA was used as a model protein. HT-29 and U87 cells were seeded in a chamber slide with a density of 5 ×10⁴ cells per well in 350 µL of medium and cultured for 24 h. The original medium was replaced with free FITC-BSA and FITC-BSA-loaded NPs at a final FITC concentration of approximately 1.5 µg/mL at 37 °C. After a 3 h incubation, the cells were washed three times with PBS (1×) and fixed with 4% paraformaldehyde for 10 min at room temperature, and the cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The slides were mounted with cover slips and cells were imaged with a Nikon FV1000 laser scanning confocal scanning microscope.