Live Imaging of siRNA Delivery and Intracellular Dynamics

HeLa cells stably expressing d1-eGFP or YFP-galectin-9 or transiently expressing GFP-AGO2 were plated in microscopy slides as described. Cells were transferred to a preheated microscopy incubation chamber, and 4–6 positions with sparse and evenly distributed cells were selected. Immediately before starting image acquisition, lipoplexes formulated with siGFP-1, siGFP-2, siLuc, or siRNA-AF647(as) were added dropwise to the medium. Typically, 5 or 10 µL of the siRNA-lipoplex solution was added to the well. For d1-eGFP knockdown experiments, one well was left untreated as a control and imaged using the same settings as treated cells. Two z-plane images with 4 µm z-spacing were acquired per position at 5 min intervals. The lower z-plane was set in the lower third of the cells (see confocal microscopy section for details). AF647-siRNA fluorescence was detected with an Airyscan detector while Hoechst 33342 and d1-eGFP fluorescence was detected with a PMT detector. Typically, images were acquired for 12–32 h for knockdown experiments and 6–8 h for galectin-9 recruitment experiments. AF647-siRNA bleaching was quantified in non-internalized glass-adhering lipoplexes.
For high-temporal resolution imaging of cytosolic siRNA release, a single z-plane set in the lower third of the cell was acquired at 5 s intervals (single position) and typically imaged for 25 min.