Visualizing Intracellular Trafficking of dsRNA

Since CypHer5E has a fluorescence maximum around pH 5 and almost no fluorescence around pH 7, it can indicate localization within the late endosomes.^{35 (link)} One day prior to the experiment, 1.2 × 10⁵ cells/well were seeded in eight well chamber microscope slides (NuncTM Lab-TekTM II, ThermoFisher Scientific, Waltham, MA). The next day, 25 ng of CypHer5E-dsGFP, either naked or conjugated with PLR10 (PLR:dsRNA mass ratio of 1:1), c-PLR10/Au/BSA and c-PLR10/Au/Hyd NPs (NP:dsRNA mass ratio of 5:1) and washed one time with DI water to remove free CypHer5E-dsGFP were mixed with 100 μL of SF900 II SFM medium then added to cells. At 4 h after exposure, the cells were washed twice with 1X phosphate-buffered saline (PBS), fixed with 100 μL of 4% paraformaldehyde for 20 min at room temperature and stained with EverBrite mounting medium (Biotium, Fremont, CA) containing 4′,6-diamidino-2-phenylindole (DAPI). The cells were visualized with Leica SP8 confocal microscope at 63× magnification by overlapping DAPI (Blue; for nuclei), Alexa 633 (Red; for CypHer5E labeled dsRNA), and bright-field (BF) filters images.