Sarkosyl-insoluble tau fibrils were prepared from FAD-ReN (ReN-mAP (enriched)) cells, which were differentiated in 3D for 7 weeks28,29. 3D-cultured cell pellets were homogenized in 1 volume of TBS extraction buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM NaVO3, 1 mM NaF, a protease inhibitor mixture (Roche Molecular Biochemicals), a phosphatase inhibitor cocktail (Thermo Scientific), 2 mM PNT (EMD Millipore) and 1 mM PMSF (Sigma-Aldrich) by using battery-operated spinning homogenizer (MIDSCI). TBS homogenates were then mixed and homogenized with 1 volume of 2x RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% w/v sodium deoxycholate, 2% v/v NP-40, a protease inhibitor mixture (Roche Molecular Biochemicals), a phosphatase inhibitor cocktail (Thermo Scientific), 1 mM NaF, 1 mM NaVO3, 2 mM PNT (EMD Millipore) and 1 mM PMSF (Sigma-Aldrich), incubated on ice for 15 min. After centrifugation (18,000 × g for 20 min at 4°C), 20% w/v N-Lauroylsarcosine (sarkosyl) stock solution was added to the RIPA-soluble supernatant fraction to adjust the final sarkosyl concentration into 1%. After incubating at room temperature with gentle rocking for 1
