Image analysis suite

To test the ability of Alveofact-coated polyplexes to cross the mucus layer secreted by 16HBE14o- cells, cells were transfected with Alveofact-coated polyplexes at different Alveofact:PEI ratios (0, 2.5:1, 5:1) containing 100 pmol AF647-siRNA and incubated for 24 h at 37°C and 5% CO₂. Once the incubation time was completed, AF488-wheat germ agglutinin was added to the cells and incubated for 15 min at 37°C and 5% CO₂ to stain the mucus layer. Afterwards, cells were washed 2 times with PBS and the membrane was cut and mounted on glass slides using FluorSave™ reagent. Membranes were immediately analyzed with a SP8 inverted confocal laser scanning microscope (Leica Camera, Wetzlar, Germany). The images were exported from the Leica Image Analysis Suite and processed with the Fiji distribution of ImageJ.

To evaluate the cellular uptake of Alveofact-coated polyplexes in ALI culture, amine-modified siRNA was labelled with succynimidyl ester (NHS) modified AlexaFluo647 dye according to the manufacturer’s protocol and subsequently purified via ethanol purification as previously reported [26 (link)].
Differentiated 16HBE14o- cells were transfected with polyplexes prepared at different Alveofact:PEI coating ratios (0, 2.5:1, 5:1) with 100 pmol AF647-siRNA and incubated for 24 h at 37°C and 5% CO₂. Afterwards, cells fixed in 4% PFA for 15 min, washed 3 times with PBS and permeabilized with PBS + 0.3% Tween20 for 10 min. Cytoskeleton was then stained by incubation with rhodamine phalloidin for 60 min, followed by nuclei staining with 0.5 μg/ml solution of 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. The membrane was then cut and mounted using FluorSave™ reagent on a glass slide and analyzed with an SP8 inverted confocal scanning microscope (Leica Camera, Wetzlar, Germany). The images were exported from the Leica Image Analysis Suite and processed with the Fiji distribution of ImageJ.