Dmem high glucose

The SH‐SY5Y human neuroblastoma cell line (ATCC^® CRL‐2266^TM) was purchased from the American Type Culture Collection. Cell culture and differentiation were performed as described previously (Tagai et al., 2020 (link)). In brief, SH‐SY5Y cells were cultured in Dulbecco's modified eagle medium (D‐MEM)/Ham's F‐12 (FUJIFILM Wako Pure Chemical Corporation, Cat#048‐29785) containing 10% fetal bovine serum (FBS), 100‐units/ml penicillin, and 100‐μg/ml streptomycin in a 37°C incubator in an atmosphere of 5% CO₂ and 100% relative humidity. In the cell differentiation, SH‐SY5Y cells were inoculated at an initial density of 1.0 × 10⁵ cells/dish in a collagen I‐coated φ 3.5 cm dishes. All‐trans‐RA (FUJIFILM Wako) was added 2 days after plating at a final concentration of 10 μM dissolved in high‐glucose D‐MEM (FUJIFILM Wako, Cat#043‐30085), which was supplemented with 15% FBS. After 5 days of culturing in the presence of RA, cells were washed with high‐glucose D‐MEM and incubated for 2 days with 100 ng/ml human recombinant BDNF (FUJIFILM Wako) in high‐glucose D‐MEM without L‐glutamine and phenol red (FUJIFILM Wako, Cat#040‐30095) containing 4 mM sodium pyruvate and 1 mM L‐glutamine. We prepared ndSH‐SY5Y cells using a modified differentiation protocol according to previously described methods (Agholme et al., 2010 (link); Encinas et al., 2000 (link); Krishtal et al., 2017 (link)).

Primary culture of mouse cerebellar astrocytes (25 (link), 26 (link)) was prepared using C57BL/6 pregnant mice (Japan SLC). Cerebella from postnatal day one of pups were dissected then digested in Hank’s balanced salt solution (Wako) containing 2.5% trypsin (Wako). Cerebellar tissue was incubated at 37°C for 30 min with continuous shaking. Mixed cerebellar tissue was centrifuge at 3500 rpm for 15 minutes at 4°C, the supernatant was discarded, and cells were resuspended in an astrocyte culture medium (high-glucose DMEM (Wako), 10% heat-inactivated FBS, and 1% penicillin/streptomycin). Cells were counted and 10–15 × 10⁶ cells were plated on Collagen I coated 10–cm dishes (Iwaki, Japan), then incubated at 37°C in a CO₂ incubator. The astrocyte culture medium was replaced with PBS on day 3 in vitro (DIV3). Oligodendrocyte precursor cells were removed by shaken the dishes for 2–5 minutes. Discard the supernatant then replaced with a fresh astrocyte culture medium. On DIV7, astrocytes were harvested and then plated on Collagen I coated 6 or 24 well dishes (Iwaki). Cells were then used for co-culture studies.

HeLa cells were cultured in minimum essential medium (MEM, Gibco, 11095072, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, CCP-FBS-BR-500, Cosmo Bio, Tokyo, Japan) and 1% penicillin-streptomycin (10,000 U/mL, Thermo Fisher Scientific, Waltham, MA, USA) for 3 days. The adherence of HEK-293 cells to the Petri dish was relatively weak; hence, the glass Petri dish was coated with fibronectin before seeding cells. For HEK-293 cells, glass Petri dishes were first coated with fibronectin and placed in humidified atmosphere incubator with 5% CO₂ at 37 °C. After coating the dish with fibronectin, HEK-293 cells were cultured. Both HEK-293 and SAOS-2 cells were cultured using high glucose DMEM (FUJIFILM Wako Pure Chemical Co., Ltd., Tokyo, Japan) with 10% FBS and 1% penicillin–streptomycin (PS).