Notch is required for the induction of functionally tolerogenic DC by MSC. A two-stage proliferation assay was carried out. Stage one: BALB/c DC (0.5 × 106/ml) were pulsed with OVA (20 μg/ml) in the presence or absence of allogeneic MSC (1.5 × 105/ml), GSI (1 μM) or DMSO (vehicle control) for 24 hours. Stage two: DC were harvested and placed into a second proliferation assay with DO11.10 CD4+ T cells (1:4, 1 × 105 DC: 4 × 105/ml T cells for 72 hours. Proliferation was analysed using thymidine (5 μCi/ml) incorporation quantified as mean counts per minute (± SEM) by liquid scintillation. Data represent three studies (*, P < 0.05; **, P < 0.01). DC, dendritic cells; DMSO, dimethyl sulfoxide; GSI, gamma secretase inhibitor; MSC, mesenchymal stem cells; OVA, ovalbumin; SEM, standard error of the mean.