Assay of Inflammasome Activation

To activate NLRP3 inflammasome, cells were primed with LPS (0.25 μg/mL) for 3 hours, followed by treatments with ATP (2 mM, 45 minutes) or nigericin (5 μM, 45 minutes). To stimulate absent in melanoma 2 (AIM2) inflammasome, cells were transfected with poly dA:dT (1 μg/mL) using a Lipofectamine 2000 (Invitrogen) for the indicated times according to the manufacturer’s protocol. Inflammasome activation was determined by the detection of active caspase-1 p20 and active IL-1β in culture supernatants using immunoblots and by quantification of extracellular IL-1β using the mouse IL-1β Quantikine ELISA kit (R&D Systems). To assess the presence of active caspase-1 in the brain, hippocampal slices were prepared from mouse brains and dissociated into single cells as described above. Cells were then stained with an active caspase-1-specific FAM-FLICA (ImmunoChemistry Technologies) probe in accordance with the manufacturer’s protocol. FLICA-positive cells were then assayed via flow cytometry.