Sh30066

Co-culture experiments were carried out using exECs or DCs and thymocytes harvested from mechanically dissociated thymus from untreated mice, or in the case of DC analysis whole thymus cultures were used. Harvested thymocytes were incubated with either 100 nM dexamethasone (D2915, Sigma Aldrich, Germany), or 20 μM z-VAD-FMK (2163, Tocris, UK) for 4 hours at 37°C prior to co-culture, washed twice with PBS, and resuspended in exEC media for co-culture (1 × 10⁶ cells / well). Cells were harvested 20 hours post co-culture and prepared for either qPCR analysis or flow cytometry analysis. Thymic DCs were isolated from untreated mice using CD11c UltraPure microbeads (130–108-338, Miltenyi Biotech, USA), on enzymatically digested thymuses. DCs were cultured in DMEM (11965, GIBCO), 10% FBS (SH30066.03, HyClone, GE Life Sciences), and 1% PenStrep (15240–062, Invitrogen). For TAM receptor inhibitor studies, exECs were treated with 25 μM RXDX-106 (CEP-40783, s8570, Selleck Chemicals) 30 minutes prior to incubation with dexamethasone treated or z-VAD-FMK treated thymocytes, and Bmp4 expression was determined by qPCR analysis 20 h post co-culture. HEK293 cells (ATCC, Manassas, VA) were cultured in DMEM (11965, GIBCO), 10% FBS (SH30066.03, HyClone, GE Life Sciences), 1% Glutamax (35050061, Life Technologies), and 1% PenStrep (15240–062, Invitrogen).