Sodium selenate

Analytical grade sodium chloride (NaCl), sodium sulfate (Na₂SO₄) potassium sulfate (K₂SO₄), sodium dicarbonate (NaHCO₃), calcium chloride (CaCl₂), magnesium chloride (MgCl₂), cadmium acetate (Cd(CH₃COO)₂), lead acetate (Pb(CH₃COO)₂), nickel chloride (NiCl₂), copper chloride (CuCl₂), potassium acid pyroantimonate (K₂H₂Sb₂O₇.4H₂O), sodium selenate (Na₂SeO₄), ammonium dimolybdate ((NH₄)₂Mo₂O₇), sodium borohydride (NaBH₄, 98%), dithionite (Na₂S₂O₄), and ferric chloride hexahydrate (FeCl₃.6H₂O) were purchased from Sigma-Aldrich (Shanghai, China). 250 ml glass (GG-17) and plastic (poly(4-methyl-1-pentene), PMP) beaker were purchased from sinopharm. All chemicals were used without further purification. Deionized water was used for all reagent and particle suspension preparation.

Neuroblastoma SHSY-5Y cells (ATCC CRL-2266, a kind gift from Dr. El-Agnaf, QBRI) were prepared and differentiated as described by Encinas et al. [50 (link),70 (link)]. In brief, cells were plated 10,000 cells/2 mL of media (Dulbecco’s modified Eagle’s medium (DMEM) containing heat-inactivated bovine fetal serum 12% (F12), 5 mL glutamax and penicillin (20 units/mL), streptomycin (20 mg/mL). All trans-retinoic acid (RA) was added to the media at a final concentration of 10 µM (50% ethanol v/v) all material from Thermo Fisher Scientific, (Waltham, MA, USA). Cells were maintained at 37 °C in a saturated humid atmosphere containing 95% air and 5% CO₂. After 5 days in the presence of RA, cells were washed three times with DMEM and incubated 24 h with sodium selenate (Sigma-Aldrich, St. Louis, MO, USA), dissolved in media to the required concentration (0, 1, 3, 5 and 10 µM).

All animal procedures were approved by the Swiss cantonal veterinary office. Primary glial cells were dissociated from cortex of males and females Wistar Han rat pups at 3-days postnatal as previously described.^{34 (link)} Cells were cultured in DMEM, 10% fetal calf serum (FCS), 1% penicillin-streptomycin at 37 °C, 5% CO₂. After 7 days in vitro (DIV), cells were infected with lentiviruses (multiplicity of infection: 5) to overexpress GCLC shRNA: GGAGGCTACTTCTATATTA or scrambled shRNA: CTTACAATCAGACTGGCGA. Puromycin was added 48 h post-infection (Calbiochem, 1ug/mL). After 10 DIV, oligodendrocytes progenitor cells (OPCs) were separated from astrocytes and microglia by overnight shaking. OPCs were plated at 1.2,10⁵ cells/well in 12-wells plate (DMEM, 2.5 uM forskoline (Calbiochem), 50 ug/ml apotransferrin, 5 ug/ml insulin (Sigma), 30 nM sodium selenate (Sigma), 10 nM hydrocortisone (Sigma), 10 nM D-biotine (Sigma), 1 mg/ml bovine serum albumin (Sigma), 10 ng/ml PDGF-AA (Sigma), 10ng/ml human fibroblast growth factor-basic (Sigma)); experiments were performed 14 DIV. In parallel, astrocytes which remained attached after shaking were plated at 7.5,10⁴ cells/well in 12-wells plates in normal culture media (DMEM, 10% FCS, 1% penicillin-streptomycin) or in differentiation media (DMEM, 2% FCS, 15uM di-butyryl cyclic AMP (Enzo) for 8 days).