Anti mouse cd4 magnetic beads

To detect the expression of phosphatidylserine (PS) in EVs, EVs recovered were labeled with Annexin‐V‐FITC (BD Biosciences, San Jose, CA, USA) for 15 min. After rinsing, all labeled EVs were detected using FCM on the CytExpert Flow Analyzer. Pacific blue 450 nm laser channel was used to circle the cell size. In addition, CD4⁺ naïve T (1.5 × 10⁵ cells/well) isolated by positive selection with anti‐mouse CD4 magnetic beads (R&D Systems) were collected and further cultured with EGFP⁺DC‐derived EVs (secreted by 1.2 × 10⁷ cells) for 48 h in 100 μl of T cell medium per well (96‐well plate), and analyzed by FCM. Cells were stained for the surface marker anti‐CD4‐APC (Biolegend, San Diego, CA, USA). After rinsing, all labeled cells were detected using FCM on the CytExpert Flow Analyzer. Data were analyzed with CytExpert.

CD4⁺ naïve T (1.5 × 10⁵ cells/well) isolated by positive selection with anti‐mouse CD4 magnetic beads (R&D Systems) were collected and further cultured with different stimuli‐induced DC‐derived EVs (secreted by 1.2 × 10⁷ cells) for 6 d in 100 μl of T cell medium per well (96‐well plate), and analyzed by FCM. For intracellular cytokine staining, cells were stimulated for 4 h with 1 μg/ml PMA (Sigma‐Aldrich) and 1 μg/ml ionomycin (Calbiochem, Germany) in the presence of 10 μg/ml brefeldin A (Sigma‐Aldrich). Cells were stained for the surface marker anti‐CD4‐V450 (BD Bioscience), anti‐CD4‐FITC (eBioscience, Waltham, MA, USA), and anti‐CD25‐APC (Biolegend). Cells were then fixed, permeabilized and labelled with intracellular and intranuclear staining reagents according to the manufacturer's instructions (eBioscience), and further stained with anti‐IL‐17A‐phycoerythrin (PE)‐cyanine (Cy7), anti‐forkhead box P (Foxp)3‐PE (Biolegend), anti‐IFN‐γ‐APC, or anti‐IL‐13‐PE (Thermo Fisher Scientific). All labeled cells were detected using FCM on the CytExpert Flow Analyzer. Data were analyzed with FlowJo software.