2.10 Endocytosis pathway of RS-NC
The endocytosis pathway of the RS-NC was revealed using flow cytometry and endocytosis inhibitors. First, 2.0 × 105MDA-MB-231 cells were seeded to wells of a 6-well plate. After 18-hour incubation at standard conditions, 10 μg/mL of chlorpromazine, 5 mM of Methyl-β-cyclodextrin, 1 μM of Cytochalasin D, and 1 μg/mL Filipin III were added to each well, respectively, and incubated for 30 minutes. Then, RS-NC (N/P ratio 5) with 200 nM FITC-labeled siRNA was added to each well and incubated for 4 hours at standard conditions. After rinsing cells with DPBS three times, cells were detached using 0.25% Trypsin 200 μL for 2 minutes. After 1 mL DMEM addition, cells were centrifuged at 300×g for 5 minutes. Finally, dispersed cells were loaded to the flow cytometry for analysis.