Assays

Post-nuclear supernatants were prepared from testis of wild type and SPPL2c-/- mice (n=5 per genotype). After removal of the tunica albuginea, testicles from two wild type mice were minced with an ultraturrax in 250 mM sucrose, 10 mM HEPES-NaOH, pH 7.4 and 1 mM EDTA (HB, homogenisation buffer) and then further homogenised by eight strokes of a Potter homogeniser. For sedimentation of nuclei, the tissue homogenate was centrifuged at 750 x g for 10 min. The supernatants were collected as post-nuclear ...

Samples for mass spectrometry were obtained from 8- to 9-week-old, male mice. Mitochondria were immunopurified from cerebellum according to the described protocol with the alteration that the final mitochondrial pellet was washed twice in IB without EDTA and BSA. Sample were lysed in 100 µL SDT lysis buffer (4% w:v SDS, 100 mM DTT in 100 mM Tris-HCl pH 7.6) by heating for 5 min at 95°C and ultrasonication (Vialtweeter: 6 times for 30 s, 100% Amplitude, 50% cycle, max power; Hielscher Ultrasonics). ...

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 ...

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 ...

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 ...