2.10. Endocytosis pathway study of complex
In a 6-well plate, HeLa cells were seeded at a density of 4.0×105 cells per well. After replacing the medium with Opti-MEM™, cells were treated with each inhibitor for 30 minutes. Inhibitors were Chlorpromazine (10 μg/mL), Methyl-β-cyclodextrin (5 mM), Cytochalasin D (1 μM), and Filipin III (1 μg/mL) (Dutta and Donaldson, 2012). The complex of final 200 nM Cy3-labeled siRNA and FITC-labeled S-R15 was self-assembled as described above. After adding 325 μL of complex to inhibitor-treated cells, cells were incubated for 4 hours at 37°C in a 5% CO2 incubator. Cells with the fluorescence of Cy3 and FITC were analyzed using flow cytometry, as mentioned in 2.6.