Horse serum

PC12 cells (a gift from Rolf Heumann, Bochum, Germany; similar to clone 251 (Heumann et al., 1983 (link))) were cultured in DMEM with high (4.5 g/l) glucose (PAN biotech) supplemented with 10% horse serum (Biochrom), 5% fetal calf serum (Biochrom) and 100 U/ml penicillin/100 ng/ml streptomycin (PAN biotech). During the course of the project, characteristic features of the cell line — such as morphology, expression of neuronal proteins, and their responsiveness to NGF — were regularly confirmed. Cells were maintained at 37°C and 5% CO₂ in a sterile incubator and tested negative for mycoplasmic infections (GATC Biotech, Konstanz, Germany).
Cells were transfected with the Neon Transfection System (Thermo Fisher Scientific, Waltham, MA, USA). The tip (100 µl) was loaded with 10 µg plasmid DNA of each construct. Cells were transfected by applying a pulse at 1410 V and 30 ms pulse width. Cells were plated onto poly-L-lysine (PLL) (Sigma, Cat. No: P-1524) coated coverslips (25 mm diameter, Menzel Gläser, Braunschweig, Germany)) and maintained for at least 48 hr before imaging.
For membrane sheet generation, cells were subjected to a brief ultrasound pulse in ice-cold sonication buffer (120 mM KGlu, 20 mM KAc, 20 mM HEPES-KOH, 10 mM EGTA; pH 7.2).

HEK293T cells were used for testing the functionality of the different vectors described below. Cells were cultured under normal oxygen conditions in a 5% CO₂ humidified incubator at 37°C. Growth medium consisted of Dulbecco's modified eagle medium (DMEM, Gibco, Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (FBS; Lonza BioWhittaker, Basel, Switzerland) and 1% Penicillin/Streptomycin (Gibco).
Murine MSCs from C57Bl/6 mice were obtained from the lab of Prof. dr. D Prockop, Tulane University, USA [21] (link). Cells were cultured in a 5% CO₂ humidified incubator under normoxic conditions at 37°C in growth medium containing Iscove's Modified Dulbecco's Medium (IMDM, Gibco), 10% FBS, 10% horse serum (Biochrom, Berlin, Germany), 1% L-glutamine (Gibco) and 1% Penicillin/Streptomycin.

The sections were first treated with citrate buffer (0.1 M citric acid, 0.1 M sodium citrate), then blocked for 20 min with 3% H₂O₂ followed by a 30 min incubation step with horse serum (Biochrom GmbH, Berlin, Germany). Myogenin mouse-monoclonal antibody (MyBioSource/Biozol MBS438443) was incubated overnight at 4 °C. The sections were first labeled with a biotinylated linker, followed by streptavidin-conjugated horseradish peroxidase, and finally counterstained with hematoxylin.

The cell line Oli-neu was cultured in modified Sato media [28 (link)]. Cell culture dishes were coated with poly-L-lysine (Sigma). HEK293T cells were cultured in DMEM (Sigma) with 10% Horse serum (Biochrom) and 1% Sodium-pyruvate (Sigma). Transfection of HEK293T cells with the NG2del constructs was effected by a standard protocol using the GenePulserXcell (Bio-Rad). Transcription was increased by including 4 mM sodium butyrate for the protease assay.
Cerebella of postnatal day 8–9 homozygous NG2-EYFP(NG2-KO) mice [29 (link)] or C57BL/6N mice (as control) were dissociated in 1% trypsin, 0.05% DNase in HBSS using a fire-polished Pasteur pipette to obtain a single-cell suspension, followed by seeding on poly-L-lysine-coated dishes. The cells were cultured in B27 medium containing DMEM, pyruvate, triiodo-L-thyronine, L-thyroxine (Sigma), B27 supplement (Gibco), 10ng/ml PDGF, 5ng/ml FGF (PrepoTech) and 1% HS. The medium was changed on the following day and renewed every 3–4 d. After 10-14d (after morphological assessment) cultures were stressed with H₂O₂ in B27 medium without growth factors.
Glioblastoma cells (R10) were cultured on ECM (Sigma) gel-coated dishes. ECM Gel was diluted 1/10 with Neurobasal media. Growth medium was Neurobasal medium (Invitrogen) with N2 supplement, B27 supplement, L-glutamine, EGF, FGF and 1% [v/v] penicillin/streptomycin (Serva).

MCF-10A cells (Cat.No. CRL-10317, ATCC) were cultured in DMEM/Ham’s F12 medium containing l-glutamine (Cat.No. FG 4815, Biochrom) supplemented with 5% horse serum (Cat.No. 12449C, SAFC), 20 ng/ml human epidermal growth factor (Cat.No. E9644, Sigma-Aldrich), 10 μg/ml insulin (Cat. No.I9278, Sigma-Aldrich), 100 ng/ml cholera toxin (Cat.No. C8052, Sigma-Aldrich), 500 ng/ml hydrocortisone (Cat.No. H0888, Sigma-Aldrich) and 100 U/ml penicillin/streptomycin (Cat.No. A 2213, Biochrom).
MDA-MB-231 and MDA-MB-436 cells were cultured in DMEM containing 4.5 g/l glucose, l-glutamine (Cat.No. FG 0435, Biochrom) supplemented with 10% fetal bovine serum (Cat.No. S 0615, Biochrom) and 100 U/ml penicillin/streptomycin.
All cell lines were incubated at 37 °C in a 95% air and 5% CO2 atmosphere. The culture medium was changed every 2 to 3 days and cells were passaged every 4 to 5 days. To detach the cells, a PBS solution containing 0.025%(w/v) trypsin and 0.011%(w/v) EDTA (Cat.No. L 2113, Biochrom) was applied for several minutes.