Fig. 2.

TF/FVIIa signalling augments cytokine-induced beta cell death independently of downstream coagulation. MIN-6 cells were pre-treated with FVIIa (10 nmol/l), FXa (100 nmol/l) or FVIIa+hirudin (10 U/ml) for 6 h, followed by treatment with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h. (a) Cell death was quantified using a cell-death ELISA by measuring absorbance (A₄₀₅–A₄₉₀). (b) Cells were stained with PI and Hoechst 33 258 (HO), ~2,000 cells/group were counted and the number of PI-positive cells was determined by fluorescence microscopy. (c) MIN-6 cells were first pre-treated with FVIIa (10 nmol/l) for 6 h. Indicated groups were then treated with IL-1 Ra (500 ng/ml) for 30 min followed by treatment with individual cytokines or with cytokine mixture for 72 h. Cell death was quantified using a cell-death ELISA by measuring absorbance (A₄₀₅–A₄₉₀). Results are means ± SEM from four experiments. *p < 0.05 for indicated comparisons, using paired Student’s t test