Nunclon sphera 96 well u bottom plate

WT or CD44^−/− CT26 (4T1) cells were seeded in a Nunclon Sphera 96 well U-bottom plate (Thermo Fisher) at a density of 10000 cells per well with 6 µg/ml of collagen I to form a single spheroid per well^{83 (link)}. After 96 h incubation, dense spheroids were formed, which were confirmed by microscopic examination. The cell spheroids were then incubated with Cy5.5-labeled PCL, PCL/CS or PCL-CP NPs, with or without pre-treatment with chlorpromazine (6 µg/ml) for 2 h. After 18 h incubation with NPs, the spheroids were gently rinsed by saline 3 times. The penetration ability was observed by a confocal laser scanning microscope with Z stack scanning (CLSM, FluoView 3000, Olympus) at 30 μm intervals from the bottom to the middle of the spheroids.

WT or ITGA5 KO LLC cells were seeded in a Nunclon Sphera 96 well U‐bottom plate (Thermofisher) at a density of 10 000 cells per well with 6 µg mL⁻¹ of collagen I to form a single spheroid per well.^[35 (link)
^] After 96 h incubation, dense spheroids were formed, which were confirmed by microscope. The cell spheroids were incubated with rhodamine‐labeled PVD or PAZA NPs (500 ng mL⁻¹ rhodamine) respectively, with or without pretreatment with transcytosis inhibitor 6 µg mL⁻¹ chlorpromazine (6 µg mL⁻¹) for 2 h. In a separate study, the cell spheroids were incubated with rhodamine‐labeled PVD or PAZA NPs (500 ng/mL rhodamine) in 10% FBS medium, blank medium, or medium with fibronectin (10 µg mL⁻¹) respectively. After 18 h incubation with NPs, the cells were gently rinsed by saline for three times. The penetration ability was observed by a confocal laser scanning microscope with Z stack scanning (CLSM, FluoView 3000, Olympus) at 20 µm intervals from the bottom to the middle of the spheroids.

HNSCC cell lines (5000 cells/well) were cultured on Thermo Scientific™ Nunclon™ Sphera™ 96-well U-bottom plates for 7 days in complete medium. Spheroid formation was observed using light microscopy (Olympus FSX-100 microscope equipped with the Olympus cellSens platform). Spheroids were treated for 48 h with each QMT or with cisplatin. After 48 h, cell death was evaluated with the two-color LIVE/DEAD^® Viability/Cytotoxicity Kit (Thermo Fisher Scientific^®). Briefly, a solution containing the green-fluorescent calcein-AM (stains live cells) and the red-fluorescent ethidium homodimer-1 (stains dead cells) was added to each well. Following 2 h of incubation with this solution images were analyzed using the FSX-100 microscope (Olympus^®).