Islets from non-diabetic human donors were treated with proinflammatory cytokines (1000 IU/mL IFN-γ, 50 IU/mL IL-1β) and 10 μM ML355 for 24 h, then lysed in lysis buffer (Thermo Fisher) and subjected to electrophoresis on a 4%–20% gradient SDS–polyacrylamide gel. Antibodies included mouse monoclonal antibody directed against PD-L1 (1:1000; Cell Signaling) and mouse monoclonal antibody directed against β-actin (1:1000; Cell Signaling). Immunoblots were visualized using fluorescently labeled secondary antibodies (LI-COR Biosciences) and were quantified using LI-COR software.
Islets were obtained from wildtype and Alox15−/− mice and treated with a proinflammatory cytokine cocktail containing 25 ng/mL IL-1β (Prospec), 50 ng/mL TNF-α (Prospec), and 100 ng/mL IFN-γ (Prospec) for 18 h. Islets were then transferred to a glass-bottom culture dish (MatTek) and islet death was measured using the Live/Dead Viability/Cytotoxicity kit (Invitrogen). Images were acquired using a MVX10 microscope (Olympus) and quantification was completed using ImageJ.
Islets were obtained from wildtype and Alox15−/− mice and treated with a proinflammatory cytokine cocktail containing 25 ng/mL IL-1β (Prospec), 50 ng/mL TNF-α (Prospec), and 100 ng/mL IFN-γ (Prospec) for 18 h. Islets were then transferred to a glass-bottom culture dish (MatTek) and islet death was measured using the Live/Dead Viability/Cytotoxicity kit (Invitrogen). Images were acquired using a MVX10 microscope (Olympus) and quantification was completed using ImageJ.