Cell Culture

The human Ntera2/D1 cell line (NT2, RRID: CVCL_3407) was obtained from the American Type Culture Collection, VA, USA. Cells were maintained and cultivated in DMEM/F12 culture medium (Invitrogen, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Invitrogen), and 1% penicillin/streptomycin (Invitrogen) in an atmosphere of 5% CO₂ at 37 °C. Using a differentiation protocol in free-floating aggregates (Paquet-Durand et al. 2003), 95% pure postmitotic neurons could be generated within 28 days. Neuronal differentiation and precursor cell migration are measured in 96-well plates on days 9, 11, or 16, respectively (Fig. 1). Each plate contained a dilution series of seven concentrations with six technical replicates of the same concentration per experiment, and each experiment was performed three times on different passages of NT2 precursor cells. For each test compound, relevant concentrations were determined in a range finding experiment with log 10 dilutions (data not shown), before deciding on the final range of concentrations used.

Three DNT endpoints assessed on NT2 cultures in vitro. (a) Neuronal differentiation was assessed by measuring immunofluorescence of a monoclonal antibody against b-tubulin type III. On average, ~ 10% of all DAPI-positive cells (blue) display ß-tub III-immunoreactivity (red) after 9 days in culture under control conditions. (b) NT2 precursor cell migration was measured using the Oris cell migration assay, which creates cell culture monolayers with a circular hole. During 2 days in culture, cells migrate a distance of ~ 400 µm on average into this hole. (c) Neurite growth was assessed by cultivating dissociated NT2 cultures after 2 weeks exposure to retinoic acid (containing ~ 20–40% neurons) and measuring ß-tubulin type III labeled neurites (red) after 24 h. Green lines: example measurements of two neurites, 46 µm and 76 µm long. Scale bars: 50 µm (a, c), 400 µm (b)