Assays

The exosome pellets were lysed in 80 µL of a modified RIPA lysis buffer (50 mM TrisHCl pH 8, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v)) with protease inhibitors (Sigma Aldrich, US) on ice with intermediate vortexing. 20 µL H2O, 10 µL 100 mM MgCl2, and 25 units Benzonase (Sigma Aldrich, US) were added followed by an incubation for 30 min at 37°C at 1400 rpm in a Thermomixer (Eppendorf, Germany). Undissolved material was removed by centrifugation for ...

The neurons were lysed direct on the plate in RIPA lysis buffer using a cell scraper. The lysate was transferred into a fresh Eppendorf tube and undissolved material was removed by centrifugation for 5 min at 20,000 g and 4°C. A protein assay was performed and 15 µg of protein were subjected to proteolytic digestion with the SP3 protocol. Proteins were reduced by addition of 9 µL of 200 mM dithiothreitol (Biozol, Germany) in 50 mM ammonium bicarbonate and incubation for 30 min at 37°C. Cysteine ...

LUHMES cell culture LUHMES cells were cultured and differentiated as described previously (Sholz et al., 2011). 0.5 Million undifferentiated LUHMES (unLUHMES) cells were seeded into a Poly-D-Lysine coated 6-well containing growth media (DMEM F12, 1% N2 Supplement, 0.04 µg/mL bFGF) and harvested at day three in vitro. The live cell count was around 1 Million cells using Trypan blue and an automated cell counter (Biorad). For differentiation, 1 Million unLUHMES were seeded into a Poly-D-Lysine ...

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 ...

Samples were prepared by in solution digestions. For details, see the methods part of the accompanying paper.

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 ...