Culture plates

Animal studies were approved by the School of Biology Ethics Committee at the University of St Andrews. C57BL/6 mice were bred as needed. Mouse primary hippocampal and cortical neurons were cultured from postnatal Day 0–1 pups. Brains were dissected in cold DMEM and dissociated with 0.05% Trypsin (Gibco, Paisley, UK) at 37 °C for 15 min prior to trituration. Cortical neurons were passed through a 40 μm cell strainer (Corning Falcon, Durham, NC, USA) and centrifuged for 5 min at 180× g. The pellet was resuspended in neuron culture medium (Neurobasal A (Gibco, Grand Island, NY, USA) supplemented with 1× B-27 (Gibco, Grand Island, NY, USA), 1× GlutaMAX (Gibco, Paisley, UK), 1× Penicillin-Streptomycin) and seeded on poly-D-lysine (Gibco, Carlsbad, CA, USA) coated culture plates (ThermoFisher Scientific, Rochester, NY, USA), coverslips (VWR, Lutterworth, UK), or Lab-Tek chambered coverglasses (ThermoFisher Scientific, Rochester, NY, USA) at appropriate densities for the intended studies. To enrich the culture for neurons and suppress glial cell proliferation, neurons were treated with 1 μM 5-Fluoro-2′deoxyuridine (FdU, Cayman Chemical, Ann Arbor, Michigan) on DIV 4. Neurons were transduced on DIV 6–8. Neurons were cultured for 10–21 days and treated as indicated. Aβ treatment was performed in Neurobasal A supplemented with 0.5× B-27 as previously described [33 (link)].

Canine recombinant IL-1β and NGAL were purchased from Kingfisher Biotech, Inc. (Saint Paul, MN) and United States Biological (Salem, MA), respectively. TRIzol, anti-zonaoccludin-1 (ZO-1) mouse monoclonal antibody (Clone: ZO-1-1A12), Alexa Fluor 488-conjugated F(ab′)2 fragments of goat anti-rabbit IgG (H+L), Alexa Fluor 594-conjugated F(ab′)2 fragments of goat anti-mouse IgG (H+L), TO-PRO-3-iodide, and ProLong Gold Antifade Reagent were purchased from Life Technologies Co. (Carlsbad, CA). PrimeScript RT Master Mix and SYBR Premix Ex Taq II were obtained from TaKaRa Bio Inc. (Shiga, Japan). Rabbit monoclonal antibodies against E-cadherin (Clone: 24E10) were purchased from Cell Signaling Technology Japan (Tokyo, Japan). The mitogen-activated protein kinase (MAPK) inhibitors FR180204, SB239063, SP600125, and U0126, and the IκB kinase inhibitors BAY-117082 and 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) were purchased from Sigma-Aldrich Inc. (St Louis, MO). The NGAL assay kit was purchased from BioPorto Diagnostics A/S (Hellerup, Denmark). StatMate IV was purchased from ATMS (Tokyo, Japan). Culture plates, dishes, and flasks were obtained from Thermo Fisher Scientific, Inc. (St. Waltham, MA).

Extracted deciduous and wisdom teeth were obtained and checked for the viability of their pulp. The extracted teeth surfaces were cleaned and then cut up by sterilized hammers and cracked open to extract the dental pulp. Afterwards, the pulp was digested in a solution of 3 mg/ml collagenase type I for 0.5 h at 37 °C, and then treated by trypsin for 2 min. Cell suspensions were seeded in culture plates (Thermo, USA) and cultured in DMEM with 10% FBS (Hyclone, USA). After cells reached a concentration of 80%, they were cultured under separate conditions: in the E8 group, the cell suspensions were incubated at a cell density of 2 × 10⁵ cells per culture plate in albumin-free E8 culture medium (Stemcell, USA) containing chemically defined and concentration-determined multiple factors; in the SCM group, the cell suspensions were plated at the same cell density in normal medium containing DMEM and 5% FBS as a control. Passage (P)3 cells were employed for the following analyses.