CD98 siRNA (m)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 4.0×10^4.	siRNA concentration-100 nM

Upstream tips
Seed 4.0×10^4.
Protocol tips
siRNA concentration-100 nM

Publication protocol

Colon-26 cells and RAW 264.7 macrophages were seeded in eight-chamber tissue culture glass slide (BD Falcon) at a respective density of 4.0×104 and 3×103 cells/well and incubated overnight. The culture medium was exchanged to serum-free medium containing scCD98-functionalized FITC-siRNA (Life Technologies)-loaded NPs (weight ratio, 40:20:1). The FITC-siRNA concentration in the medium is set as 100 nM. After 6 h of incubation, the cells (Colon-26 cells and RAW 264.7 macrophages) were thoroughly rinsed with cold PBS to eliminate excess of NPs, and then fixed in 4% paraformaldehyde for 20 min. To observe cellular uptake of NPs, DAPI (Invitrogen) was diluted 10,000 times and added for staining nucleus for 5 min. BMDMs was tested for cellular uptake at different time points (0, 1, 3 and 6 h), and Oligofectamine reagent (Invitrogen) was used as control siRNA carrier. To observe lysosomal escape of NPs, cells were subsequently stained with 50 nM LysoTracker Red (Invitrogen) for 30 min and DAPI for 5 min. Images were acquired using an Olympus equipped with a Hamamatsu Digital Camera ORCA-03G.

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