Neurobrew

For rat primary OPC cell culture preparation brains were collected from neonatal rats (3–7 day). The tissue was incubated in 1 mL Accutase and 4 μL DNase/mL (Sigma, St Louis, MO) at +37 °C and passed through a fire–polished Pasteur pipette every 10 min, this being repeated three times with decreasing pipette guage. The homogenate was directly labeled and isolated using magnetic anti-A2B5 beads (130-093-388) (Milteny Biotec, Bergisch Gladbach, Germany) instead of Percoll layering as for flow cytometry. Throughout the study, culture ware was pre-coated with Poly-l-lysine (Sigma, St Louis, MO) at +37 °C for >2 h, rinsed with distilled water twice and incubated with Fibronectin (Sigma, St Louis, MO) at +37 °C for >1 h. Cells were cultured in 200 μL Neurobrew (Milteny Biotec, Bergisch Gladbach, Germany), 100 μL N2-supplement (Thermo Fisher Scientific, Waltham, MA), 2 μL bFGF (PeproTech, Princeton, NJ) and 2 μL PDGF-BB (R&D Systems, Minneapolis, MA) per 10 mL of DMEM/F-12 media (Thermo Fisher Scientific, Waltham, MA). For differentiation bFGF and PDGF-BB was removed from the media. Cells were proliferated for 5–7 day at +37 °C and 5% CO₂ in a humidified incubator, harvested with Accutase and re-seeded; 50 000cells/well for 96-well plate (Nunc, Rochester, NY) or 75-100 000cells/well for staining in chambers (177402PK) (Nunc, Rochester, NY).

HEK293 cells and stable HEK(pTRAF^{Nrf2/HIF/NFkB}) reporter cells were cultured in Eagle’s minimum essential medium, supplemented with 10% fetal bovine serum and 100 U penicillin/ml and 100 μg streptomycin/ml. Cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂ and 21% O₂. Primary rat cultures were established from neonatal pups. Microglia and oligodendrocytes were isolated as described for flow cytometry; following Percoll layering, cells were labeled with either anti-cd11b or anti-A2B5 Microbeads (Milteny Biotec, Bergisch, D). Microglia were cultured in DMEM/F12 supplemented with 100 U penicillin/ml and 100 μg streptomycin/ml and 5% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA). Oligodendrocytes were cultured in in 200 mL Neurobrew, 100 mL N2-supplement (Milteny Biotec, Bergisch, D), 2 mL bFGF (PeproTech, Princeton, NJ), and 2 mL PDGF-BB (R&D Systems, Minneapolis, MA) per 10 mL of DMEM/F-12 media on poly-l-lysine pre-coated culture ware. For differentiation of oligodendrocytes, bFGF and PDGF-BB was removed 48 h prior to stimulation.

Primary cortical neurons were prepared from embryonic 14–15-day fetal mice (C57BL/6J, male and female, wild type) (CLEA Japan, Tokyo, Japan). Cortical tissues dissected from the brain were treated with trypsin (0.25%, Nacalai Tesque, Kyoto, Japan) for 20 min in Hank’s balanced solution, and neurons were gently dissociated. The obtained neurons were plated on poly-ethylenimine (0.1%, Sigma) coated glass bottom dishes at a density of 2–5 × 10⁴ cells/dish. Neurons were cultured in MEM culture medium (Nissui, Tokyo, Japan), supplemented with l-alanyl-l-glutamine (0.5 mM, Nacalai Tesque), fetal bovine serum (2%), NeuroBrew (2%, Miltenyi Biotec, Tokyo, Japan), HEPES (10 mM, pH7.4), and glucose (4.5 g/L).
To investigate the thermal effect on neurite extension in neurons, the polymers (FPT_High and control copolymer) were introduced into neurons with a solution of iso-osmotic glucose solution (5%) for 5 min at 4 °C. The shape of the cells, including the protrusions was visualized by the fluorescence of the loaded polymers, and the length of the neurites was measured using Fiji software.