Dmem low glucose

Bovine serum albumin (BSA)‐conjugated PA, 2‐PG, and 3‐PG were prepared as previously described [26 (link)]. Briefly, 500 mmol·L⁻¹ of each fatty acid/monoacylglycerol was dissolved in ethanol at 55 °C. Meanwhile, 10% free fatty acid (FFA)‐free BSA was dissolved in Dulbecco's modified Eagle's medium (DMEM) containing 1.0 g·L⁻¹ glucose (DMEM low glucose; NACALAI TESQUE, Kyoto, Japan) at 37 °C and subsequently filtered through Millex‐GV Syringe Filter with a 0.22 μm pore size hydrophilic polyvinylidene difluoride membrane (Merck Millipore, Burlington, USA). Each of the dissolved fatty acid/monoacylglycerols was mixed with 10% FFA‐free BSA in a 1 : 100 ratio, vortexed, and incubated at 55 °C for 15 min, followed by a 15 min conjugation in a water sonicator, until the fatty acid/monoacylglycerol‐BSA was dispersed. The vehicle control was prepared by mixing ethanol and 10% FFA‐free BSA in a 1 : 100 ratio. Fatty acid/monoacylglycerol‐BSA and the vehicle control were stored at −20 °C and melted in a water bath at 55 °C prior to use; thereafter, they were diluted in DMEM low glucose (NACALAI TESQUE) with 10% fetal bovine serum (Biowest, Nuaille, France) and 1% of Penicillin–Streptomycin‐Amphotericin B suspension (Wako, Odawara, Japan) to 100 or 120 μmol·L⁻¹. The vehicle control was diluted in the same manner as the fatty acid/monoacylglycerols.

Ceramide species (16:0, 24:0, 24:1) and GlcCer species (16:0, 24:1) were from Avanti Polar Lipids (Alabaster, AL, USA). GlcCer (24:0), LacCer (16:0, 24:0, 24:1), and GM3 (16:0, 18:0, 20:0, 22:0, 24:0, h24:0, 24:1) and GM1 (18:0) were synthesized as described previously (Murase et al, 1989; Mauri et al, 1999). GM2 (from brain of Tay‐Sachs disease patient) was from Matreya (State College, PA, USA). Brain GD1a, GD1b, and GT1b were from Sigma‐Aldrich (St. Louis, MO, USA). Brain GQ1b was from AdipoGen Life Sciences (San Diego, CA, USA). Milk GD3 was from Nagara Science Co. (Gifu, Japan). Ceramides, GlcCer, and LacCer species were dissolved at 1 mM in warmed DMSO. Gangliosides were dissolved at 0.5 mM concentration in warmed low‐glucose DMEM (Nacalai Tesque; Kyoto, Japan). Stock solutions were stored at −30°C and diluted with low‐glucose DMEM to 100 μM concentration prior to experiments.

Vero cells were cultured in low-glucose DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 2% fetal calf serum (FCS). 293T cells were cultured in high-glucose DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FCS. Human iPSCs 110 (BYS0110), 112 (BYS0112), and 116 (BYS0116), were obtained from ATCC (Manassas, VA, USA). BMI and BM9 were obtained from WiCell (Madison, WI, USA). 201B7 and 409B2 were obtained from the Riken Bioresource Center (Ibaraki, Japan). Epi was obtained from Thermo Fisher Scientific. Epi, BMI, BM9, 110, 112, and 116 were maintained on Corning Matrigel hESC-Qualified Matrix (CORNING, Corning, NY, USA) in mTeSR 1 medium (STEMCELL Technologies, Vancouver, BC, Canada). 201B7 and 409B2 were cultured on vitronectin (Thermo Fisher Scientific) in ReproFF2 medium (ReproCELL, Kanagawa, Japan) supplemented with 5 ng/mL basic fibroblast growth factor (bFGF; ReproCELL). T-705, Y27632, and puromycin were purchased from Selleckchem (Houston, TX, USA), FUJIFILM Wako Pure Chemical (Osaka, Japan), and InvivoGen (San Diego, CA, USA), respectively.