Glutamax supplement

MEFs were cultured on plates treated with a 0.1% gelatin solution (20 min on 37 °C). MEF culturing medium consisted of high-glucose DMEM, 10% FBS, 1X NEAA, 1X penicillin–streptomycin, 1X GlutaMAX^TM supplement (Gibco-Invitrogen, Waltham, MA, USA) and 0.1 mM β-mercaptoethanol. Induced pluripotent stem cells (iPSCs) were cultured on feeder cells plated onto gelatin-coated plates. iPSC medium consisted of high-glucose DMEM (Gibco-Invitrogen, Waltham, MA, USA), 15% KnockOut™ Serum Replacement (Gibco-Invitrogen, Waltham, MA, USA), 1X NEAA (Gibco-Invitrogen, Waltham, MA, USA), 1X penicillin–streptomycin (Gibco-Invitrogen, Waltham, MA, USA), 1X GlutaMAX^TM supplement (Gibco-Invitrogen, Waltham, MA, USA), 0.1 mM β-mercaptoethanol (Gibco-Invitrogen, Waltham, MA, USA) and 10 ng/mL mouse leukemia inhibitory factor (LIF Recombinant Mouse Protein, embryonic stem cell-qualified, Gibco-Invitrogen, Waltham, MA, USA). All cell types used were tested for mycoplasma.

We cultured cells of the spiral ganglia from CD-1 mice or sorted PANs/SGNNCs from Tau-EGFP mice on glass bottom dishes coated with 0.2% gelatin in “astro-medium” containing DMEM/Ham's F12 with GlutaMAX™ supplement with 10% heat inactivated FBS (Invitrogen), 5% heat inactivated horse serum, 2% B27 supplement (Thermo Fischer Scientific), 10 ng/ml EGF (Sigma-Aldrich), 10 ng/ml bFGF (Invitrogen), and 6.0 mg/ml of D-(+)-glucose (Heinrich et al., 2010 (link)). At 60–70% confluency, we transfected cells with the expression vector(s). Cells were transfected using Lipofectamine LTX (Life Technologies) based on previously published methods (Heinrich et al., 2010 (link)) and manufacturer's instructions. We incubated the cells in DNA/Lipofectamine complexes for 4 h at 37°C with 5% CO₂. After replacing DNA/Lipofectamine complexes with astro-medium and incubating for 20 h, the cells were maintained in “differentiation medium” consisting of DMEM/Ham's F12 with GlutaMAX™ supplement, 2% B27 supplement, 1% N2 supplement, 10 ng/ml of BDNF (Peprotech), 10 ng/ml of NT-3 (Peprotech) and 6.0 mg/ml of D-(+)-glucose (Oshima et al., 2007 (link)) for an additional 6 to 10 days.

When the cells attained a confluence of 40%, mTeSR™1 was replaced with RPMI 1640 (Gibco), containing 2% B27 minus insulin (Gibco), 100 ng/mL activin A (Peprotech), and 50 ng/mL Wnt 3a (R&D System) for 3 days. The medium was then replaced with IMDM (Procell) with 20% knockout serum replacement (Gibco), 1% GlutaMAX™ supplement (Gibco), 1% dimethyl sulfoxide (DMSO) (Sigma-Aldrich), 1% nonessential amino acids (NEAA) (Gibco), and 0.1 mM β-mercaptoethanol (β-ME) (Sigma-Aldrich) for 4 days. Next, the cultures were placed in IMDM containing 1% GlutaMAX™ supplement, 20 ng/mL oncostatin M (OSM) (Peprotech), 5 ng/mL basic fibroblast growth factor (bFGF) (Peprotech), 0.5 μM dexamethasone (Dex) (Sigma-Aldrich), and 1% insulin-transferrin-selenium (ITS) (Sigma-Aldrich) for 5 days. Finally, the differentiated cells were cultured in hepatocyte culture medium (HCM) (Lonza) supplemented with 10 ng/mL hepatocyte growth factor (HGF) (Peprotech), 0.5 μM Dex, 10 μM lithocholine acid (LCA) (Sigma-Aldrich), 10 μM vitamin K₂ (Sigma-Aldrich), and 1% ITS for continuous cultivation.

K562 and THP1 cells were cultured in filter sterilized RPMI 1640 (Gibco, 21870-076) containing 10% heat-inactivated, low-endotoxin FBS (Gibco, catalog no. 10082139) and 2 mM GlutaMAX supplement (Gibco, catalog no. 35050061). HeLa cells (ATCC CCL2) were cultured in filter-sterilized Dulbecco modified Eagle medium–F-12 (Gibco, catalog no. 11320033) with 2 mM GlutaMAX supplement and 10% fetal bovine serum (FBS).

All chemicals were prepared as concentrated solutions according to the recommendations of the different manufacturers. Compounds were aliquoted in Eppendorf tubes and used once, to avoid repeated freezing/thawing processes. Aliquots were stored at −80 °C for no longer than two months. Care was taken to protect photosensitive molecules from light by wrapping the test tubes in aluminum foil. Drugs were extemporaneously diluted at their respective final concentration in DMEM containing 10% FBS+ N2+ B27 minus Anti-Oxidant (AO). In order to study the impact of Glucose on Rotenone induced toxicity, two distinct DMEM formulations differing only in their glucose concentration were used. HG conditions correspond to DMEM, high glucose, Glutamax Supplement, Pyruvate (Thermofisher, Gibco, Cat ref 10569010) containing 4.5 g.L⁻¹ (25 mM) of glucose. LG condition corresponds to DMEM, low glucose, Glutamax Supplement, Pyruvate (Thermofisher, Gibco Cat ref 10567014) containing 1 g.L⁻¹ (5 mM) of glucose. Ten days after seeding, Rotenone (5 μM, Sigma-Aldrich) with or without mdivi-1 (20 μM, Tocris) or SP 600125 (10 μM) diluted in LG or HG DMEM were applied for 24 or 48 h according to the treatment. After treatment, neurons were fixed with Paraformaldehyde 4% (Sigma-Aldrich) for 10 minutes at room temperature then washed with PBS.

Brain organoids were generated from hESCs as previously described 39 (link), but slightly modified. Briefly, for embryoid body (EB) formation, hESCs were washed twice with DPBS, incubated with Accutase for 5 minutes, and dissociated into single cells. 3000 single cells were seeded in each well of low attachment 96-well U-bottom plate in E8 medium containing 10 μM ROCK inhibitor and centrifuged at 100 g for 3 min, then medium was half changed every other day. On day 4, EBs were transferred to low attachment 24-well plate in neural induction medium containing DMEM-F12 (GIBCO) with 1% N2 supplement (GIBCO), 1% Glutamax supplement (GIBCO), 1% MEM-NEAA (GIBCO) and 1 μg/mL Heparin (GIBCO), and medium was changed after 48 h. On day 7, EBs were transferred into Matrigel droplets as previously described and cultured in brain organoid differentiation media containing 50% DMEM-F12, 50% Neurobasal, 200x N2 supplement, 0.025% Insulin (GIBCO), 100x Glutamax supplement, 200x MEM-NEAA, 100x penicillin-streptomycin, 0.035% 2-Mercaptoethanol and 100x B27 supplement without Vitamin A, and medium was changed after 48 h. On day 10, organoids were transferred to orbital shaker (Corning) in brain organoid differentiation media with Vitamin A, medium was changed every 4 days.

Normal human epidermal
keratinocytes (nHEK) were seeded into a 96-well plate (167008, Thermo
Fisher) for 5 × 10³ cells per well on a monolayer
of 3T3 fibroblasts (7 × 10³ cells/well), previously
treated with mitomycin C (4 μg/mL, M4287, Sigma-Aldrich), and
cultured in 100 μL of FAD standard medium [FAD medium (custom-made,
Gibco) containg 1× GlutaMAX supplement (35050061, Gibco), 5 ng/mL
insulin, 10 nM cholera toxin, 0.5 μg/mL hydrocortisone, 10 ng/mL
epidermal growth factor, and 10% fetal calf serum (FCS)] in a humid
chamber at 37 °C and 5% CO₂. After 50–60% confluency
was reached, cells were treated with CNF from 1000 to 1 μg/mL
in FAD standard medium containing 1% FCS for 24 h. Untreated and 0.1%
(v/v) Triton X-100-treated cells were used as 100 and 0% viability
controls, respectively. After 24 h, the medium was then replaced with
Dulbecco’s modified Eagle’s medium (DMEM) phenol free
(31053-028, Gibco) containing 1% FCS, 1×X GlutaMAX supplement,
and 10% Alamar Blue (DAL1100, Invitrogen) solution. Cells were continued
to culture for 3 h before the fluorescence was read at 560 nm of excitation
and 590 nm of emission in a SpectraMax iD3 fluorometer. The cell viability
(%) was calculated as (test sample – blank)/(untreated control
– blank) × 100%. Three independent replicates were carried
out for each assay. GraphPad Prism was used for statistics and data
analysis.