Notch engagement by Jag1 clusters on the nanoscale suggests a force-independent mode of activation
Notch engagement by Jag1 clusters on the nanoscale suggests a force-independent mode of activation
Cells were collected using Trypsin‐EDTA (0.025%, Thermo Fisher Scientific). AF22 Neuroepithelial Stem cells (NES) were cultured as adherent cells on cell culture flasks previously coated with 20 μg/ml polyornithine (Sigma) for 1 hour and 1 μg/ml Laminin2020 for 4 hours (Sigma). Cells were cultured on NES culture medium contained DMEM/F12+GlutaMax (Gibco), supplemented with 10 μl/ml N‐2‐supplement (100×, Thermo Fisher Scientific), 10 μl/ ml Penicillin‐Streptomycin (10,000 U/ml, Thermo Fisher Scientific), 1 μl/ml B27‐supplement (50×, Thermo Fisher Scientific), 10 ng/ml of bFGF (Life Technologies) and 10 ng/ ml of FGF (PeproTech). The culture medium was replaced every second day. The NES cells were passaged enzymatically when reaching 100% confluency using Trypsin‐EDTA (0.025%, Thermo Fisher Scientific). NES cells were seeded at a density of 40,000 cells/cm2. Nes cells were seeded at the density of 18750 cell/cm2 and let inside the cell culture incubator to attach for 6 hours. Cells were stimulated with different Jag1Fc nanopatterns for 3 hours. RNA extraction was performed by using RNeasy Micro Kit (Qiagen). Libraries were prepared with TruSeq stranded mRNA (Illumina 20020595) according to manufactures instructions.