Fluorodeoxyuridine

Following washing in phosphate-buffered saline, bacteria grown in LB for 18–24 h at 37°C and standardized to an OD₆₀₀ of 3.0 were prepared for tests. To obtain a synchronously growing population, eggs were prepared by treating a population of C. elegans with hypochlorite/NaOH solution and transferring the resulting eggs to NGM plates covered with E. coli OP50, as previously described (Chen et al., 2014b (link)). The synchronized adult L4 worms on NGM plates were washed in M9 buffer. After centrifugation, the pellets of worms were re-expanded with S medium, and 5 μl of solution containing approximately 30–40 worms were placed in each lawn of 48-well plates with 5 μl fluorodeoxyuridine (Sigma–Aldrich, Saint Louis, MO, USA) to prevent reproduction. Finally, 190 μl of bacteria in LB solution were added to achieve a total 200 μl in each lawn. Assay plates were incubated at 25°C for 3–6 days. The percentages of animal death were calculated as the numbers of dead animals/total animals found each day under a dissecting microscope.

(-)-Epicatechin (EC), 2´-7´dichlorofluorescein diacetate (DCFH-DA), ampicillin sodium salt, nistatine, agar, yeast extract, fluorodeoxyuridine (FUdR), phosphate-buffered saline (PBS), cholesterol, Bradford reagent, guanidine hydrochloride (GuHCl), 2,4-dinitrophenylhydrazine (DNPH), malondialdehyde, hexanal, hexenal and 4-HNE were purchased from Sigma-Aldrich (Madrid, Spain). Dimethyl sulfoxide (DMSO) was obtained from Panreac (Barcelona, Spain) and trichloroAcetic acid from Fluka Analytical (Madrid, Spain). HPLC grade acetonitrile was from Carlo Erba (Rodano, Italy). Acetic acid was from Merck (Darmstadt, Germany). Fluorescein thiosemicarbazide was from Carbosynth (Berkshire. UK)

Quercetin (Q), ampicillin sodium salt, nistatine, agar, yeast extract, fluorodeoxyuridine (FUdR), phosphate-buffered saline (PBS), cholesterol, and 2-mercaptoethanol, were purchased from Sigma-Aldrich (Madrid, Spain). Dimethyl sulfoxide (DMSO) was obtained from Panreac (Barcelona, Spain). SYBR^® SelectMaster Mix and high-capacity cDNA reverse transcription kit were from Applied Biosystems (Carlsbad, CA, USA), and the Illustra™ RNAspin mini isolation kit was from GE Healthcare (Buckinghamshare, UK).

Dorsal root ganglia of embryonic day 15 rats were dissociated with TrypLE Express and collected by centrifugation (250× g, 5 min, 4 °C) [16 (link),17 (link),18 (link),19 (link),20 ,21 (link)]. Cells were resuspended in neuron maintenance medium comprising Neurobasal medium (Thermo Fisher Scientific) supplemented with 2% B27 and NGF (20 ng/mL, Millipore, Burlington, MA, USA) and then seeded at 100,000 cells/cm² onto poly-d-lysine/laminin-coated plates. Endogenous Schwann cells and fibroblasts were eliminated following brief treatment with fluorodeoxyuridine and uridine (10 μM each, Sigma-Aldrich), whereas dorsal root ganglion neurons and neurite network remained adherent on the coated plates.
aMSC-derived OPs were seeded at 10,000 cells/cm² onto the axonal network of dorsal root ganglion neurons and the co-culture was maintained in myelination medium comprising Neurobasal medium supplemented with B27, NGF neutralizing antibody (5 μg/mL, Abcam, Cambridge, UK), thyroid hormone (10 ng/mL, Sigma-Aldrich), and DAPT (10 ng/mL, Sigma) for 14 days. Myelin-forming OLs and axons in co-cultures were immunostained for myelin basic protein (MBP) and neurofilament 200 (NF200) respectively. Co-cultures were also prepared for examination of compact myelin under transmission electron microscopy. aMSCs in co-culture with dorsal root ganglion neurons were prepared in parallel as controls.

Hippocampal organotypic cultures were prepared from postnatal day 5 (P5) C57BL/6 wild-type (WT) mice (derived from the colony at the TU Braunschweig) as previously described [21 (link)]. Neonatal mice were decapitated before the hippocampi were dissected in ice-cold sterile Gey’s balanced salt solution (GBSS) and sliced transversally at a thickness of 400μm using a McIllwain tissue chopper. The slices were placed on Millicells CM membrane inserts (Millipore) and cultivated in a 37°C, 5% CO₂, 99% humidity environment in a medium containing 50% BME (Eagle, with Hanks salts without glutamine), 25% HBSS, 1ml of glucose (50%), 25% donor equine serum (HyClone), and 0.5 ml of L-glutamine (200 mM stock solution) for 100ml. To reduce the number of non-neuronal cells, a mixture of antimitotic drugs (cytosine arabinoside, uridine, and fluorodeoxyuridine; 10^–6–10^–7 M each; Sigma-Aldrich) was applied for 24h.

Explants were cultured as previously described (Fueshko and Wray, 1994 (link); Klenke and Taylor-Burds, 2012 (link)). Briefly, embryos were removed from time-pregnant NIH-Swiss at E11.5, the nasal region containing the olfactory pits was dissected and placed in Gey’s balanced salt solution (Life Technologies, Inc.) supplemented with glucose (Sigma Chemical Co.). The sex of the embryos was not determined. Each embryo generated one nasal explant. Nasal explants were adhered to coverslips by a chicken plasma (Cocalico Biologicals)/Thrombin (Sigma) clot and maintained at 37°C in serum-free media (SFM) in a humidified atmosphere with 5% CO₂. The medium was changed after 3 days and supplemented with one dose of fluorodeoxyuridine (4–10 μM; Sigma) to inhibit proliferation of dividing olfactory neurons and non-neuronal tissue (e.g., fibroblasts).