Doxycycline hydrochloride

Neuronal induction by doxycycline-inducible NGN2 expression was performed as previously described^{27 (link)}. Briefly, on day 0, iPSCs were dissociated with TrypLE Select (GIBCO, Thermo Fisher Scientific, MA, USA) and disseminated on a mixed coating of poly-l-lysine (final 0.0002% v/w, Merck, Darmstadt, Germany), and Matrigel (final 2% v/v, Corning, NY, USA). The disseminated iPSCs were cultured in Neurobasal Medium (GIBCO, Thermo Fisher Scientific, MA, USA) supplemented with 0.5% B27 without vitamin A (GIBCO, Thermo Fisher Scientific, MA, USA), 1 × Glutamax (GIBCO, Thermo Fisher Scientific, MA, USA), 2 mg/mL doxycycline hydrochloride (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 5 mM Y-27632 (Nacalai-Tesque, Kyoto, Japan). Y-27632 was withdrawn on day 1 or 2. On day 5, we exchanged culture media for new media without doxycycline hydrochloride. To evaluate the amount of Aβ between timepoints, on day 8, all culture media were replaced with fresh medium. On days 10, 12 and 14, we recovered old media as samples for the ELISA analysis. To check the effect of an oxidant (H₂O₂) or anti-oxidant (NAC), all culture media were replaced with fresh medium containing H₂O₂ or NAC on day 8. The culture media were subjected to analysis on day 10.

Tetracycline-inducible human neurogenin 2 (NGN2) construct was transduced into iPSCs using a piggyBac vector as previously described [49 (link)]. The iPSC colonies in which the construct was efficiently introduced were selected with neomycin, G418 disulfate (Nacalai Tesque). From these, subclones that could efficiently differentiate into neurons were further selected. The selected iPSCs were referred to as iN-iPSCs. For differentiation into cortical neurons, iN-iPSCs were dissociated into single cells and replated onto Matrigel (Corning Incorporated, Corning, NY)-coated plates at 300,000 cells/cm² in a neuronal medium [Neurobasal plus medium (Gibco, Thermo Fisher Scientific), 1X B27 plus (Gibco, Thermo Fisher Scientific), 1X GlutaMAX (Gibco, Thermo Fisher Scientific) and 1 × Penicillin–Streptomycin (Gibco, Thermo Fisher Scientific)], supplemented with 10 μM Y-27632 (Nacalai Tesque) and 1 μg/mL doxycycline hydrochloride (Wako Pure Chemicals Industries, Osaka, Japan). On day 5, the differentiated neurons were dissociated into single cells with TrypLE select (Gibco, Thermo Fisher Scientific) and reseeded onto Matrigel-coated 96-well plates (Corning Incorporated) at 60,000 cells/cm² in the neuronal medium with 10 μM Y-27632.