A1r a1 confocal system

The spheroids were characterized for cellular reorganization by labelling 4T1 with Calcein AM (2 μg/ml in culture media, green color) and 3T3 with Cell Trace® Calcein Red-Orange AM (4 μg/ml in culture media, pseudo-color blue) prior to spheroid formation. After 1 h incubation at 37 °C, the medium was removed and cells were washed twice with PBS. Labeled cells were utilized for spheroid formation, as described above. The resulting intact and live spheroids were imaged using confocal laser scanning microscope (Nikon A1R-A1 confocal system, 10× objective) after 24 and 48 h.

Homospheroids of 4T1 tumor cells and heterospheroids formed with different ratios of 4T1 and 3T3 cells were incubated with 1.25 μg/μl of Cy5-PNPs suspended in serum-free DMEM medium. Spheroids were incubated with Cy5-PNPs in microwelled-Petri dishes in a humidified incubator at 37 °C with 5% CO 2 . After exposure to Cy5-PNPs, 3 intact spheroids per time point were washed with PBS and subsequently mounted in PBS for confocal imaging. Confocal images were taken using a confocal laser scanning microscopy (Nikon A1R-A1 confocal system, 10× objective) at 1, 24, and 48 h to observe the penetration of the PLGA nanoparticles into intact and live spheroids. Calcein green fluorescence representing 4T1 tumor cells was excited using 488 nm laser line. Pseudo-color blue of calcein red-orange fluorescence, representing NIH3T3 cells, was visualized with 575 nm laser excitation. Red fluorescence of Cy5 dye, representing PLGA NPs conjugated to Cy5 dye, was excited with 638 nm laser line. Subsequently, Cy5-PNPs penetration were quantified digitally using NIH ImageJ software.