2.5 Fluorescence imaging of NCs
Cellular uptake of NCs was observed using fluorescence imaging. First, 1 × 104 MDA-MB-231 cells were seeded to wells of a 24-well plate and incubated for 16 hours in standard conditions. 200 nM Cy3-labeled siRNAs were incubated with R11, RS, and RT peptides at the N/P ratio 20, 5, and 30 in 200 μL were treated into a total volume of 500 μL and incubated at room temperature for 30 minutes, respectively. At 4 hours after NC addition, the cells were rinsed three times with DPBS 200 μL, and the cells were fixed by 200 μL of 10% formalin solution for 10 minutes. Next, the cells were added by 200 μL of 0.1% Triton X-100 in PBS (0.1% PBST). After a 10-minute incubation, cells were added by 200 μL of 2% (w/v) BSA in 0.1% PBST and incubated at room temperature for 30 minutes. Then, the cells were incubated in the Hoechst 33342 (10 mg/ml in PBS) for 10 minutes. After rinsing cells with 200 μL of PBS twice, the cells were incubated in the Phalloidin (diluted by PBS-T as 1:200 v/v) at room temperature for one hour. After washing twice with 200 μL of DPBS, the cells were observed at 150× magnification by a fluorescence microscope (Ti-E; Nikon, Tokyo, Japan).