Exosome Labeling and Uptake

To observe the exosomal uptake by the recipient cells, Calu-3 exosomes were labeled with SYTO RNASelect green fluorescent stain and BODIPY TR ceramide red fluorescent stain. The exosomes were incubated at 37°C for 15 minutes and then purified from the excess dye using a Sepharose CL-2B 10/30 gel filtration column. As a control, PBS was mixed with dyes and purified similarly. HTBE cells were washed with PBS and treated with 1 × 10⁸ labeled Calu-3 exosomes or the same volume of SYTO RNASelect-BODIPY TR-PBS control for 1.5 hours. Cells were washed twice with PBS, fixed with 100% ice-cold methanol, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. Cells were incubated with primary mouse anti–β-tubulin IV antibody (BioGenex) overnight at 4°C and then stained with secondary donkey anti-mouse Alexa 647 antibody and Hoechst. The z-stack confocal images were acquired using an Olympus FluoView FV1000 microscope with a 60× objective. Volocity software was used to construct three-dimensional images and XZ projections.