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.