2.5. Tau Protein Labeling and In Vitro Aggregation and Uptake

The expression and purification of truncated tau was performed, as previously described [43]. The labeling of recombinant tau was performed using Alexa Fluor™ 546 Protein Labeling Kit (#A10237; Thermo-Scientific, Bratislava, Slovakia), according to the manufacturer’s instructions. The in vitro aggregation of recombinant truncated tau protein (aa151-391, based on longest tau isoform tau 40) was performed using heparin (Sigma) as an inducer at a final concentration of 25 μM in 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) overnight at 37 °C, as previously described and characterized [44]. Post incubation, the sample was centrifuged at 100,000× g for 1h at room temperature, and the pellet was re-suspended in 1× PBS, sonicated for 5 s at 20% power output using an MS72 probe of a Bandello Sonopuls Sonifier (Bandelin, Berlin, Germany), and stored as 1 μM aliquots at −80 °C. The oligomerization of the tau protein was verified by immunoblotting and electron microscopy (Supplementary Figure S2a,b). For the examination of tau uptake by astrocytes, cells were plated on 8-well Lab-Tek slides (25,000 cells/well), incubated with labeled monomeric (4 µM)/aggregated tau (1 µM), and washed and examined using LSM 710 confocal microscope (Carl-Zeiss, Jena, Germany). Images were obtained using the Zen software system (Carl-Zeiss, Jena, Germany) and fluorescent intensities were measured using Fiji (ImageJ 2.0.0). For the compartmentalization study, SiR-lysosome kit—Live cell lysosome probe (Spirochrome, Stein am Rhein, Switzerland) was used according to the manufacturer’s instructions.