Hbss 10x

Oligodendrocyte progenitor cells were isolated from post-natal day 5 (P5), P60 and P540 from Pdgfra-H2BEGFP brains^{44 (link)}, oligodendrocytes were sorted from P60 Plp-EGFP brains^{44 (link)}, using fluorescence-activated cell sorting as described previously^{38 (link)}. Tissue was dissected in HBSS 1X (HBSS 10X (Invitrogen), 0.01 M HEPES buffer, 0.75% sodium bicarbonate (Invitrogen), and 1% penicillin/streptomycin) and mechanically dissociated. After an enzymatic dissociation step using papain (30 μg/ml in DMEM-Glutamax, with 0.24 μg/ml L-cysteine and 40 μg/ml DNase I), samples were layered over a pre-formed Percoll density gradient and then centrifuged for 15 min. Cells were then collected and stained with propidium iodide (PI) for 2 min at room temperature (RT). In a second step, GFP-positive and PI-negative cells were sorted by fluorescence-activated cell sorting (FACS; Aria, Becton Dickinson) and collected in pure fetal bovine serum. For RNA/DNA extraction, cells were washed twice in PBS 1X (PBS 10X, Invitrogen), then the dry cell pellets were frozen at −80 °C. For immunocytochemistry, cells were washed once in PBS 1X (PBS 10X, Invitrogen), then plated and maintained in Bottenstein and Sato medium (without growth or differentiation factors)^{38 (link)} for 12 h before fixation.

Primary hippocampal neurons were obtained from E18 wild-type mice embryos. Hippocampi collected were stored in HBSS 1× (10 mL HBSS 10X, GIBCO; 3.30 mL HEPES 0.3 M at pH 7.3; 1 mL PenStrep, Sigma Aldrich, Milan, Italy; 87 mL sterile water) and then chemically digested with 0.5% trypsin in HBSS 1× for 15 min at 37 °C. The tissues were gently dissociated with a sterile micropipette to isolate the cells. Neurons were plated on 24 mm slides previously coated with poly-L-lysine at the density of 150 k cell for each slide. Neuronal cultures were maintained in culture medium (100 mL Neurobasal, GIBCO; 2 mL B27 Supplement 50X, GIBCO; 1 mL PenStrep, Sigma Aldrich, Milan, Italy; 250 μL Glutamine 200 nM, GIBCO; 125 μL Glutamate 10 mM, Sigma Aldrich, Milan, Italy), in incubator at the constant temperature of 37 °C and in the presence of 5% CO₂.

Acid chloride (1 N)
Ethanol (70% [v/v] in distilled water)
Calcium chloride solution (Ca Cl₂) (1 M; filtrated on a 0.22 μm filter)
Citric acid solution 1 N
Formaldehyde (4% [w/v] in distilled water)
Hanks balanced salt solution (HBSS 10x) (Gibco 14185-052)
Hepes solution (kept at 4 °C) (1 M; filtrated on a 0.22 μm filter)
Hydrochloric acid solution 1 N
Isopropanol (kept at 4 °C)
Magnesium chloride solution (Cl₂Mg) (1 M; filtrated on a 0.22 μm filter)
Milliq water (filtrated on a 0.22 μm filter)
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
Sodium chloride solution 2 M (kept at 4 °C)
Sodium citrate (5% and 10% [w/v] in distilled water. Adjusted pH 7) (kept at 4 °C)