Low endotoxin fetal bovine serum

Murine mesangial cells (MCs) were kindly provided by Dr. Jian Yao (University of Yamanashi, Chuo, Japan) and cultured as described previously (Zhang et al., 2016 (link)). Briefly, MCs were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated low endotoxin fetal bovine serum, penicillin/streptomycin and HEPES (GIBCO, California, USA) at 37°C in a humidified atmosphere containing 95% air and 5% CO₂. MCs were treated with normal glucose (Normal, 5.5 mmol/L D-glucose) as control group, mannitol (MNT, 24.5 mmol/L MNT) as osmotic pressure control group, dimethylsulfoxide (DMSO, 0.1% DMSO) as DMSO control group and HG (30 mmol/L D-glucose) without or with HYP at the dose of 5 or 15 μg/ml and RAP at the dose of 20 nmol/L for 24, 48, or 72 h. Here, the low dose of HYP (5 μg/ml) and the high dose of HYP (15 μg/ml) were determined by the reference of Zhang et al. (2016 (link)). As HYP was dissolved in 0.1% DMSO, a similar volume of DMSO was added to DMSO control group.

Human neuroblastoma SH-SY5Y cells were maintained in DMEM/F-12 supplemented with 10% low-endotoxin fetal bovine serum, 2 mM L-glutamine, and 1% penicillin-streptomycin (all from Gibco, Thermo Fisher Scientific, USA). For experiments, cells were plated on clear bottom 96-well plates (Phenoplate, Perkin Elmer, USA) at 6 × 10⁴ cells/well and maintained as described in a previous study (Westbroek et al., 2016 (link)) for 24 h to allow the cells to attach. WT and GBA1 knockout (KO) immortalized neural cells were kindly provided by Ellen Sidransky from the National Institutes of Health, and the generation and characterization of these cells have been described previously (Westbroek et al., 2016 (link)). These cells were cultured in neurobasal growth media supplemented with 2% B27 supplement, 1 mM L-glutamine, and 100 U/mL penicillin-streptomycin (all from Thermo Fisher Scientific, USA) in T75 flasks (Thermo Fisher Scientific, USA) pre-coated with poly-L-lysine (Sigma-Aldrich, USA) at 37°C with 5% CO₂. For live cell imaging experiments, 2.5 × 10⁴ cells per well were plated on clear bottom 96-well plates (Phenoplate, PerkinElmer, USA) pre-coated with poly-L-lysine and incubated at 37°C with 5% CO₂ until confluent.

BV-2 cells (Saiqi, Shanghai, China), PC12 cells (Chinese Academy of Sciences, Shanghai, China), and 293T cells (Chinese Academy of Sciences, Shanghai, China) were cultured in a humidified incubator with 5% CO₂ at 37°C. The culture medium was Dulbecco's modified Eagle medium (Gibco, New York, America) supplemented with 5% low-endotoxin fetal bovine serum (Gibco, New York, America), 100 units/ml penicillin (Gibco, New York, America), and 100 μg/ml streptomycin (Gibco, New York, America).