Nucspot live 650

iPSC neurons expressing TauRD-YFP reporter were cultured as described above and plated at day 7 of differentiation onto 96-well plates, where they were allowed to mature until day 14–17 of differentiation before live-imaging experiments. Four hours before imaging, a far-red nuclear dye (NucSpot Live 650, catalog #40082, Biotium) was added to the media to allow time-lapse visualization of nuclear morphology and nuclear fragmentation associated with cell death (1:1000 in DMSO). At this time, DNAse1 (catalog #LK003170, Worthington Biochemical Corporation) was also added at a final concentration of 0.148 U/µl to predigest dead cell nuclei, which can otherwise obscure the signal from live nuclei. Within one imaging interval before the start of imaging (2 h), tau seed-containing and control brain lysates were added to the culture media at 1–10 µg of total protein into 150 µl media/well. Epifluorescent live imaging was then begun using a temperature and CO₂ controlled microscope (Cytation5, BioTek) with images taken every 2 h for 7 d. For each well imaged, a 4 × 4 or 5 × 5 grid of 10× images was acquired. Following live-imaging experiments, media was harvested, and cells were fixed for immunohistochemistry.

Preadipocytes were seeded in six well-plates and induced to differentiate into adipocytes in standard adipogenic media alone, as previously described by our group (31 (link)), in the presence of the PIEZO1 agonist Yoda1 10 µM (Tocris, Cat N° 5586), a concentration reported in different cell types (18 (link), 29 (link), 33 (link)–36 (link)). Adipogenesis was evaluated using Bodipy 493/503 (ThermoFisher Cat N° D3922), a neutral lipid staining, and the nuclear stain NucSpot^® Live 650 (Biotium, Cat N° 40082) and reported as Bodipy fluorescence intensity/nuclei count using long-term live-cell imaging IncuCyte^® S3 system. Cultured cells were imaged every 6 hours during 4 days in culture. Quantification of cell images after 4 days was performed using IncuCyte ZOOM™ software.