Exosome free medium

A PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) was utilized to label CD4⁺T cells, and a PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich) was utilized to label exosomes. CD4⁺T cells (5 × 10⁵/well) were seeded in 12-well plate(4.5 cm²/well), cultured in 500 µL exosome-free medium (UmiBio, Shanghai, China), then activated with 1 µg/mL anti-CD3 and 1 µg/mL anti-CD28 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany). Finally, they were treated with 20 µL exosomes (1 × 10¹⁰ particles/20 µL/well) for 24 hours. The uptake of labeled exosomes was detected by an inverted fluorescence microscope (LAS X3.4.7; Leica Camera, Wetzlar, Germany) and by 520-nm and 580-nm lasers. To compare the uptake of exosomes by CD4⁺T cells between patients with BU and the healthy controls, a PKH26 Red Fluorescent Cell Linker Kit was utilized to label exosomes and anti-human CD4-APC antibody (clone RPA-T4; BioLegend, San Diego, CA, USA) was used to stain CD4⁺T cells. The relative quantification of exosome uptake by CD4⁺T cells was evaluated by FCM analysis.