Assays

Blood samples (20 ml) from clinically affected homozygous Npc1 mutation carriers and healthy donors were collected. Negative selection of peripheral blood monocyte-derived macrophages was performed by incubating full blood for 20 min at RT with RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies). An equal volume of washing buffer (D-PBS/2% FBS/1 mM EDTA) was added to each sample and layer of macrophages was separated from red blood cells and plasma by centrifugation on a Ficoll ...

Primary microglia were isolated from mouse brains (cerebrum) using MACS Technology (Miltenyi Biotec) according to manufacturer´s instructions and as previously described (Daria et al., 2017). Briefly, brain cerebrum was dissected, freed from meninges and dissociated by enzymatic digestion using a Neural Tissue Dissociation Kit P (Miltenyi Biotec) and subsequent mechanical dissociation using 3 fire-polished glass Pasteur pipettes of decreasing diameter. CD11b positive microglia were magnetically ...