Fixation permeabilization buffer

Animals were injected with 200 µg BrdU (Sigma-Aldrich) i.p. After 24 h, thymi were harvested and processed to a single-cell suspension as described. For detection of BrdU incorporation, thymocytes were fixed with BD Fixation/Permeabilization buffer (BD Biosciences) for 20 min at room temperature followed by incubation with BD Cytoperm Permeabilization Buffer Plus (BD Biosciences) for 10 min on ice. Cells were then refixed with BD Fixation/Permeabilization buffer (BD Biosciences) for 5 min at room temperature. Thymocytes were treated with 30 µg DNase I for 1.5 h at 37°C to expose BrdU epitopes. Flow cytometric staining was then performed with BrdU (Bu20a).

EVs were biotinylated by incubation with 333 μM EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) for 30 min at room temperature. Excess biotins were removed by Zeba Spin Desalting Column, 7K MWCO (Thermo Fisher Scientific). Biotinylated EVs were captured on a neutravidin-coated glass slide, which was prepared according to a previously described method (Lee et al., 2018 (link)). Following 30-min incubation at room temperature, the slide was washed and further incubated with fixation/permeabilization buffer (BD Biosciences; 15 min at room temperature) and then with a blocking buffer (0.2% BSA in PBS; 20 min at room temperature). For EV labeling, a cocktail of fluorescence-labeled antibodies was introduced. Samples were incubated for 90 min at room temperature and washed with 0.2% BSA in PBS. A BX-63 upright fluorescent microscope (Olympus) with a 100X oil objective was used for imaging. All the fluorescence images were taken under the same acquisition setting (i.e., objective, exposure time, camera setting, illumination).