On day 4 (d4), NDM mixed with PAMAM dendrimers was gently replaced, incubated for 72 hours.
Growth protocol
The hNPCs were cultured in NEM (ENStem-A) supplemented with 0.05% b-FGF and 1% L-glutamine (Sigma-Aldrich, St. Louis, MO, US) on poly-ornithine-coated (Sigma-Aldrich, St. Louis, MO, US) and laminin-111-coated (Sigma-Aldrich, St. Louis, MO, US) dishes. On day 0 (d0), the cells were transferred to 96-well round bottom plates (Nunc, Falcon) at a density of 6,000 cells per well to allow neurosphere formation. On day 2 (d2), the developed neurospheres were gently transferred to a 48-well plate that was pre-coated with laminin-511 and contained neural differentiation media (Neurobasal Medium, NDM), supplemented with 1× B27, 1× N2, and 10 ng/mL BDNF (Invitrogen, Carlsbad, CA).
Extracted molecule
total RNA
Extraction protocol
RNA was prepared using the RNeasy Micro kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations. RNA was quantified using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 50ng RNA using the Low Input Quick Amp Labeling Kit (Agilent Technologies)
Hybridization protocol
600ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes following the manufacturers instructions. microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent Technologies Microarray Scanner, Scan resolution3μm.
Data processing
The scanned images were analyzed with Agilent Feature Extraction 10.7.3.1 using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
