IVIS spectrum imaging of labeled EVs

EVs isolated from cultured DC2 cells at day 11 (8 days post treatment) were labeled with XenoLight DiR Fluorescent Dye (PerkinElmer) per manufacturer’s instruction. Briefly, EV pellets were resuspended in 600 μL of cold PBS and incubated with 20 μL of 10 mM DiR dye for 5 min at RT. As a non-EV control, 600 μL of PBS alone was processed in parallel. The total volume was brought up to 38 mL with PBS and ultracentrifuged at 100,000 × g for 1 h. The dyed EV pellet was resuspended in PBS and 5 × 10⁷ particles were injected intravenously via the tail vein into naive adult F1 hybrid male mice. Twenty-four hours following injection, the mice were sacrificed and their tissues were collected for imaging using an IVIS Spectrum (PerkinElmer). The excitation filter was set at 745 nm and the emission filter was set at 800 nm. For quantification, total radiant efficiency was calculated using Living Image software, with the minimum set at 1 × 10⁷ and the maximum set at 1.45 × 10⁷. The N for IVIS experiments in Supplementary Fig. ⁵ is as follows: 6 injected Vehicle EVs, 6 injected Stress Cort EVs, where EVs were not pooled for any injection.