2.6 Flow cytometry for cellular uptake analysis
NC-mediated cellular uptake efficiency was analyzed using flow
cytometry. MDA-MB-231, MCF-7, BT-474, SK-BR-3 cells were used for breast
cancer cell types, and RAW264.7 cell was used for a normal cell type.
First, 2.5 × 104 cells were seeded to a well of a
6-well plate and incubated at 37oC for 16 hours in a 5%
CO2 incubator. Then, 200 nM FITC-labeled siRNA was
incubated with R11, RS, RT peptides of N/P ratio 20, 5,
30 in 2 mL of total volume at room temperature for 30 minutes. Next, at
4 hours at 37oC and 5% CO2 after NC
addition, cells were rinsed three times with DPBS 1 mL and detached
using 0.25% Trypsin 1 mL 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 (Cytoflex; Beckman Coulter, MA, USA) for
analysis.