2.13. Seeding assay in HEK293

HEK293 tau‐venus stably expressing human P301S 0N4R tau‐venus was described previously. ²⁷ These cells were maintained at 37°C at 5% CO₂ in complete DMEM (C‐DMEM) with 10% (vol/vol) FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The seeding assay using spinal cord homogenate from P301S or Ifnar1 ^−/− P301S‐tau mice was carried out as described previously. ²⁷ Spinal cords were homogenized using a benchtop homogenizer in 1X PBS. Briefly, HEK293 P301S tau‐venus cells were plated at 25,000 cells per well in black 96‐well plates precoated with poly D‐lysine in 50 μL OptiMEM (Thermo Fisher Scientific). Spinal cord homogenate was diluted in 50 μL OptiMEM (Thermo Fisher Scientific) and added to cells with 0.5 μL per well Lipofectamine 3000. After 1 h, 100 μL c‐DMEM was added to each well to stop the transfection process. Cells were incubated at 37°C for 48 h and subsequently fixed in 4% PFA. After staining with Hoechst, tau aggregation in the cells was quantified using high‐content image acquisition and analysis (Nikon Ti2, NiS Elements). The percentage of seeded cells was calculated by the number of tau‐venus puncta divided by the number of cells.