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.