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 ...

2 x 108 wild-type or Tspan15-knockout HEK-293T cells were lysed in 1% digitonin lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich). Lysates were pre-cleared with protein G sepharose prior to immunoprecipitation with Tspan15 mAb 1C12 chemically cross-linked to protein G sepharose with dimethyl pimelimidate (Thermo Fisher Scientific). Five independent immunoprecipitations were carried out for each cell type. Immunoprecipitation samples in non-reducing Laemmli buffer were subjected ...

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 ...

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 ...

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 ...