Assays

Microglia isolated by MACS from WT and Myd88-/- mouse pups were seeded at a density of 1×106 cells per 60-mm dish in DMEM/FCS/L929 medium. After 2 DIV, microglia were washed with warm DMEM/pyruvate medium and treated with 20 µg/mL myelin debris (or HEPES control) in 2 mL of TIC medium for 4 h. After treatment, the cells were washed with DMEM/pyruvate medium, and incubated with 4 mL of DMEM containing 0.2% BSA for 16 h. The cells in each dish were washed with 2 mL of cold PBS on ice, and lysed ...

After washing the primary cells with 1x PBS, cell-type specific growth media containing serum supplements with 50 µM of ManNAz (Thermo) was added for 48h. Afterwards, conditioned media was collected and filtered through Spin-X 0.45 µM cellulose acetate centrifuge tube filter (#8163, Costar) and stored at -20°C in protein Lobind tubes until further usage. Glycoprotein enrichment was performed using 60 µL Concanavalin A (ConA) bead slurry per sample (Sigma). ConA beads were washed twice with 1 mL ...

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 ...

Secretome analysis of primary neuronal cultures was performed using the high-performance secretome protein enrichment with click sugars" (hiSPECS) method, described in detail previously (Tüshaus et al, 2020). In brief, neurons were cultured for 48 h (DIV 5-7) in the presence of 50 µM ManNAz (#88904, ThermoFisher), cultivation media was filtered through 0.45 µm spin columns (Sigma-Aldrich, CLS8163). Glycoproteins were enriched using ConA agarose beads (Sigma, C7555) and clicked to magnetic DBCO ...

Corpus callosum dissections were lysed in 300 µL STET lysis buffer (1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, 50 mM TrisHCl pH 7.5) with a Precellys Evolution homogenizer (Bertin, Germany) using 0.5 mL soft tissue homogenization kit CK14 applying two cycles of 30 s with a speed of 6500rpm. After 15 min incubation on ice, samples were centrifuged at 16,000×g for 15 min to remove undissolved material and cell debris. The supernatant was transferred to a fresh protein lobind tube (Eppendorf, ...