ON-TARGETplus Human GAPDH (2597) siRNA - Individual

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 10^5 cells/well.	siRNA concentration-100 nM.

Upstream tips
Seed 10^5 cells/well.
Protocol tips
siRNA concentration-100 nM.

Publication protocol

Cells were seeded on 24-well plates (105 cells/well) and cultured overnight to ∼70% confluency. To quantify siRNA internalization, siRNA-polyplexes containing Cy3-labelled siRNA (Termofisher Scientific, UK) (corresponding to 100 nM siRNA) were added to the cells in serum-free medium and incubated for 5, 15, 30, 60, 120, 180 or 240 min. To establish siRNA-polyplex internalization pathways, chemical inhibitors of endocytosis were employed (Supporting Information, Fig. S1). Cells were pre-incubated with inhibitors for 1 h at a concentration at which high viability was preserved in H1299 cells (Supporting Information, Fig. S2) followed by incubation with siRNA-polyplexes in serum-free medium containing the corresponding inhibitor. Cells were then washed with PBS and harvested by trypsin. Extracellular fluorescence of polyplexes associated with the cell surface (not internalized) was quenched with 0.04% v/v Trypan blue (in PBS) and cells were analysed immediately by flow cytometry using a Beckman Coulter Altra flow cytometer equipped with 488 nm and 556 nm lasers to obtain forward and side scatter and read Cy3, respectively. The emitted fluorescence light was collected using 585/23 nm band pass filter; 10,000 cells were analysed per sample. Data was analysed using Weasel Software Version 3.0.2 (The Walter and Eliza Hall Institute of Medical Research, Melbourne Australia).

Control experiments of clathrin and caveolae inhibition studies were conducted with known ligands for the clathrin and caveolae-mediated pathways (FITC-transferrin at 100 μg/ml and cholera toxin-B-subunit at 5 μg/ml, respectively) [7] (Supporting Information, Fig. S3).

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