2.6. Cellular uptake efficiency using flow cytometry
Human cervical cancer HeLa, human dermal fibroblasts neonatal (HDFn), and immortal keratinocyte cell line HaCaT were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in 5% CO2 humidified incubator (Esco Micro Pte. Ltd., Changi, Singapore). 3.0×105 cells were added to each well of a 6-well plate and incubated at 37°C in a 5% CO2 incubator overnight. After the complex formation of the final 200 nM Cy3-labeled IL10-siRNA and peptides (20:1 N/P ratio) in serum-reduced Opti-MEM™, 325 μL of the complex was added to each well and incubated for 4 hours at 37°C in 5% CO2 incubator. The wells were washed twice with 1 mL of pre-warmed phosphate-buffered saline (PBS). After 200 μL treatment of 0.25% trypsin-EDTA for 2 minutes, 2 mL of fresh DMEM medium was added. The suspended cells were centrifuged at 360 × g for 5 minutes. After the supernatant was removed, cells were washed with PBS twice under the same conditions. The final cell pellet was resuspended in ice-cold PBS and analyzed using a flow cytometer (Gallios; Beckman Coulter, CA, USA).