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).