75 cm2 plastic culture flasks

We dissected cortico-hippocampal tissues from 16-day-old mouse embryos. For astrocytes, single cell suspension from dissociated tissues was cultured in Dulbecco’s modified Eagles medium (DMEM/F12) supplemented with 10% inactivated fetal bovine serum (FBS) (Life Technologies, Sweden) in 75 cm2 plastic culture flasks (Corning, NY, United States). Cultures were incubated at 37°C, 95% air/5% CO₂, and culture media were replaced biweekly. Inactivated astrocytes dominated cultures at 10–14 days (Cedazo-Minguez et al., 2001 (link)). For neuronal cultures, single-cell suspension from dissociated tissues was cultured in complete neurobasal media (GIBCO) following the manufacturer’s instructions and as previously described by our group (Mateos et al., 2009 (link)).

The cell culture was prepared as described previously (11 (link), 49 (link)). Human SH-SY5Y neuroblastoma cells were grown to 70%–80% confluence in 75-cm² plastic culture flasks (Corning, China) in a mixture of 5% CO₂ and 95% air at 37°C, employing Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (1:1) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin. Prior to treating SH-SY5Y cells with 300 μM zinc sulfate, the cultures were kept in free serum media. Zinc sulfate (300 μM) was chosen based on the results and protocol established in our previous study (49 (link)). SH-SY5Y neuroblastoma cells were pretreated with 20 ng/ml rapamycin for 1 h and then incubated with 300 μM zinc sulfate for 4 h.
For the generation of tau knockout cell line, the SH-SY5Y cells (5 × 10⁵) were seeded per well in a 6-well culture plate in a medium containing DMEM/F12, 10% FBS. The cells were grown until they reached 70%–80% confluence. We used SiRNA reagents (Invitrogen) to silence human Tau genes in the SH-SY5Y cells, following manufacturer’s instructions. The targeted RNA sequences that we used for Tau were: 5′CAUCCAUCAUAAACCAGGATT3′ (sense) and 5′UCCUGGUUUAUGAUGGAUGTT3′ (antisense).