2 mercapthoethanol

Lymphocytes from unaffected tissue and tumor tissue were incubated with PMA (50 ng/mL, Sigma-Aldrich), ionomycin (1 µg/mL, Sigma-Aldrich), Brefeldin A (1 µg/mL, BD), and Monesin (1,95 µM, BD) in IMDM (+ 25 mM HEPES, Gibco) with 10% FCS (Hyclone GE Healthcare), 0.1 mg/mL Gentamycin (BioWhittaker), 2 mM l-Glutamine (Merck), 50 µm 2-Mercapthoethanol (Gibco) for 5 h at 37 °C and 5% CO₂. Unstimulated cells from unaffected lamina propria served as controls. For degranulation assays, CD107a-BV421 (1D4B, BD) was added to the stimulation culture from the beginning in a 1:200 dilution.

100,000 sorted SP CD4⁺CD25⁻ thymocytes were co-cultured in 96-well round bottom plates together with 5000 thymic DCs and 10 μg of thymic exosomes (final concentration 20 × 10⁹ exosomes/ml) in 200 μl medium for 3 days. In addition, CD4⁺CD25⁻ thymocytes were cultured alone or with either exosomes or DCs. The exosome concentration used was based on dose-response experiments and earlier studies15 . To study if the exosome effects were MHC restricted, cells and exosomes from different mouse strains (C57BL/6 and Balb/c) were used in cross-strain experiments. 100 nm latex beads (Sigma-Aldrich, St- Louis, MO) were added to a final concentration of 75 × 10⁹ beads/ml to separate cultures as described for the exosomes and used as a reference for nano particle presence. The cell medium consisted of RPMI 1640 (Invitrogen, Paisley, Scotland), with 5% exosome-depleted FBS (Sigma-Aldrich, St- Louis, MO), 2 mM L-glutamine (Life Technologies), 100U penicillin/ 100 μg streptomycin (Sigma-Aldrich) and 50 μM 2-mercapthoethanol (Gibco (Life Technologies). To support thymocyte survival, IL-7 (Peprotech, Rocky Hill, NJ), 0.2 ng/ml and IL-2 (RnDSystems), 20 ng/ml, were added to the cultures.