Macs neurobrew 21

Pregnant C57BL/6J mice, (RRID:IMSR_JAX:000664) were obtained from Charles River Laboratories Japan (Yokohama, Japan). Primary cortical neurons were prepared from E15.5 pups and cultured in MACS Neuro Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with MACS NeuroBrew-21 (Miltenyi Biotec), 0.5 mM l-glutamine, penicillin, and streptomycin (all from Invitrogen) on tissue culture plates coated with poly-l-ornithine (Fujifilm, Tokyo, Japan) at a density of 5 × 10⁴ cells/cm². At 3 days in vitro (DIV), a half volume of fresh media containing 2′-deoxy-5-fluorouridine (final concentration, 3.3 µM) (Tokyo Chemical Industry, Tokyo, Japan) was added to inhibit glial proliferation. At 7 DIV, half of the media was changed, and αSyn PFF (final concentration, 10 µg/mL) with or without TA was added. αSyn PFF were incubated for 7 days and then analyzed by immunocytochemistry.

All cells were cultivated at 37 °C, 5% CO₂, 95% rel. humidity in an incubator and checked for Mycoplasma contamination monthly. MNs were differentiated from the human iPSC line IMR90-04 (IMR90), which was originally purchased from WiCell [56 (link)]. IMR90 were cultivated in StemMACS™ iPS-Brew XF medium (Miltenyi, Bergisch Gladbach, Germany) with medium changed every other day on plates that were coated with Corning^® Matrigel^® basement membrane preparation (growth factor reduced; #354230, Corning, New York, NY, USA). At 70–80% confluency, IMR90 were passaged with 0.02% EDTA in phosphate-buffered saline without Mg²⁺ and Ca²⁺ (PBS). To increase the survival of the cells, 10 µM Rho-Kinase inhibitor Y-27632 (TargetMol, Boston, USA) was supplemented after thawing and subculturing of IMR90. For neuron differentiation, neural medium with differentiation-stage specific supplements was used. Neural medium consisted of equal volumes of DMEM/F12 (#21331046, Thermo Fisher Scientific, Waltham, Massachusetts) and MACS^® Neuro medium (Miltenyi), as well as 0.5× N-2 Supplement (Thermo Fisher Scientific), 1× MACS^® NeuroBrew^®-21 (Miltenyi), 1× L-glutamine (Thermo Fisher Scientific) and 1× penicillin/streptomycin (P/S, Thermo Fisher Scientific). The protocols used for the differentiation are described in detail in Schenke et al. [28 (link)], with a brief summary given below.

Cortical cells from embryonic day 19 (E19) Sprague-Dawley rat brains (Orient Bio Inc., Seongnam-si, Korea) were cultured as previously described³⁶ . Experiments were approved beforehand by the Institutional Animal Care and Use Committee of the College of Medicine, Dongguk University (Approval no: IUCAC-2018-06). Cells were initially plated in MACS^®Neuro Medium (Miltenyi Biotec Inc., USA) supplemented with MACS NeuroBrew^®-21, 45.95 µM glutamate, 500 µM glutamine, 25 µM 2-mercaptoethanol, and 1% penicillin-streptomycin, and fed every four days with the same medium without glutamate or 2-mercaptoethanol supplementation. HEK293T cells were cultured on polylysine-coated glass coverslips in DMEM (Invitrogen) containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were transfected using Lipofectamine^® 2000 reagent (Invitrogen), and immunostained after fixing with paraformaldehyde and methanol^{37 (link)}.