2.7. Observation of cellular uptake using fluorescence microscopy
HeLa cells were seeded into a 24-well plate at the number of 1.0×105 cells per well and incubated overnight at 37°C in a 5% CO2 incubator. After the complex formation of the final 200 nM Cy3-labeled IL10-siRNA and peptides (20:1 N/P ratio), 160 μL of complexes were applied to the cells in Opti-MEM™ for 4 hours. After washing twice with 200 μL of pre-warmed PBS, the cells were fixed using 200 μL of 10% formalin solution for 10 minutes. Then, cells were serially treated with 200 μL of 0.1% Triton X-100 in PBS (0.1% PBST) for 10 minutes, then 200 μL of 2% BSA in 0.1% PBST at room temperature for 30 minutes. Cells were incubated in the Hoechst 33342 dye solution for 10 minutes under the absence of light. After washing twice with 200 μL of pre-warmed PBS, the cells were incubated in the Phalloidin dye solution at room temperature for 1 hour. After washing twice with 200 μL of pre-warmed PBS, the cells were observed at 200× magnification by a fluorescence microscope (Ti-E; Nikon, Tokyo, Japan).