SH-SY5Y human neuroblastoma cell line (ECACC, 94030304) and the mouse spontaneously immortalized microglia-9 (SIM-A9) cell line (ATCC-CRL-3265) [30 (link)] were cultured in Gibco™ DMEM/ F-12 with GlutaMAX™ (ThermoFisher, Waltham, MA, 31331028), supplemented with 10% fetal bovine serum (ThermoFisher, Waltham, MA, 10100139) and 5 μg/ml plasmocin™ (InvivoGen, San Diego, CA, ant-mpt). The culture medium of SIM-A9 cells was supplemented with 5% horse serum (Sigma-Aldrich, Saint Louis, MO, H1270). Embryonic cortical-hippocampal neurons from wild-type (WT) and SREBF-2 mice (B6;SJL-Tg(rPEPCKSREBF2)788Reh/J, RRID:IMSR_JAX:003311) were isolated on day 16–17 of pregnancy by trypsin digestion following a standard protocol [31 (link)]. Dissociated cells were grown in Neurobasal™ medium (ThermoFisher, 21103–049) supplemented with 2.5% (v/v) B27 supplement (ThermoFisher, 17504–001), 0.5 mM L-glutamine (Sigma-Aldrich, G7513) and 5 μg/ml plasmocinTM (InvivoGen, ant-mpt), and plated onto poly-D-lysine (Sigma-Aldrich, P6407)- and laminin (Sigma-Aldrich, L2020)-coated plates at a density of 2 × 105 cells/cm2. Half of the culture medium was changed every 3 or 4 days. Over 95% of neuronal purity was confirmed by immunochemistry using antibodies targeting neuronal and glial markers. Experiments were performed at 7 to 10 days in vitro (DIV). All procedures involving animals and their care were approved by the ethics committee of the University of Barcelona and were conducted in accordance with institutional guidelines in compliance with national and international laws and policies.
Cell cholesterol enrichment was achieved by incubation with a cholesterol:methyl-β-cyclodextrin complex (CHO:MCD; containing 50 μg/ml of cholesterol) (Sigma-Aldrich, C4951) for 1 h followed by 4-h recovery. To induce inflammasome activation, cells were treated with 10 μg/ml lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Sigma-Aldrich, L4391), 10 μg/ml N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate (also known as muramyl dipeptide, MDP; Sigma-Aldrich, A9519), 5 mM ATP (Sigma-Aldrich, A2383), 150 μg/ml monosodium urate crystals (MSU; Santa Cruz Biotech., sc-202711), and oligomeric Aβ at the indicated times. Preincubation with 4 mM glutathione ethyl ester (GSHee) or with the cell-permeable caspase 1 inhibitor I (10 μM; Bachem, 4095744) was performed 30 min before treatment when indicated.
Cell cholesterol enrichment was achieved by incubation with a cholesterol:methyl-β-cyclodextrin complex (CHO:MCD; containing 50 μg/ml of cholesterol) (Sigma-Aldrich, C4951) for 1 h followed by 4-h recovery. To induce inflammasome activation, cells were treated with 10 μg/ml lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Sigma-Aldrich, L4391), 10 μg/ml N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate (also known as muramyl dipeptide, MDP; Sigma-Aldrich, A9519), 5 mM ATP (Sigma-Aldrich, A2383), 150 μg/ml monosodium urate crystals (MSU; Santa Cruz Biotech., sc-202711), and oligomeric Aβ at the indicated times. Preincubation with 4 mM glutathione ethyl ester (GSHee) or with the cell-permeable caspase 1 inhibitor I (10 μM; Bachem, 4095744) was performed 30 min before treatment when indicated.
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