The in vitro myelinating cultures were either untreated or treated with human FGF9 (100ng/ml). Samples were collected at 24 hours and 10 days from the untreated and treated myelinating cultures.
Growth protocol
Neurospheres were generated from striata of P1 Sprague-Dawley rats by resuspending enzymatically dissociated cells in 20ml neurosphere media [DMEM/F12 (1:1, DMEM containing 4,500 mg/L glucose), supplemented with 0.105 % NaHCO3, 2 mM glutamine, 5,000 IU/ml penicillin, 5µg/ml streptomycin, 5.0mM HEPES, 0.0001% bovine serum albumin (all from Invitrogen), 25 µg/ml insulin, 100 µg/ml apotransferrin, 60uM putrescine, 20nM progesterone, and 30nM sodium selenite (all from Sigma)] and plating them into a 75 cm3 tissue culture flask. The culture was supplemented with 20ng/ml mouse submaxillary gland epidermal growth factor (R&D Systems) and maintained at 37ºC in a humidified atmosphere of 7% CO2/93% air. Every 2-3 days, 5 ml NSM and 4 µl EGF were added to the flask. Neurosphere-derived astrocytes (NsAs) were obtained by triturating neurospheres and plating them onto Poly-L lysine (PLL, 13ug/ml, Sigma) -coated coverslips (13 mm diameter, VWR International, Leicestershire, UK) in low glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine (Sigma) until they formed a confluent monolayer. NsAs conditioned media was obtained from confluent NsAs monolayers grown in DM without insulin in the presence or absence of 100ng/ml FGF9, medium changed every other day and stored at -80°C until further use. These supernatants were then used to treat myelinating cultures (3:1 vol/vol supernatant/fresh DM without insulin) from 18-28 DIV in the presence or absence of a neutralizing antibody specific for human FGF9 (Abcam, 10 μg/ml). Myelinating spinal cord cultures were derived from E15.5 Sprague-Dawley (SD) embryos. Spinal cords were dissected out, cleared of meninges, minced with a scalpel blade and enzymatically dissociated with 2.5% trypsin (Invitrogen) and 1.33% collagenase (ICN Pharmaceuticals, UK). Enzymatic activity was stopped by adding 1 ml of a solution containing 0.52 mg/ml soybean trypsin inhibitor, 3.0 mg/ml bovine serum albumin, and 0.04 mg/ml DNase (Sigma). The tissue was then triturated, centrifuged and resuspended in plating medium (PM, 50% DMEM, 25% horse serum, 25% HBSS) and 150,000 cells per 100 µl plated on cover slips coated with a confluent monolayer of NsAs. Coverslips were placed in 35-mm Petri dishes (3 per dish) and left in the incubator to attach for 2 hours, after which 300 µl of PM and 500 µl of differentiation medium (DM) [DMEM (4,500 mg/ml glucose), 10 ng/ml biotin, 0.5% hormone mixture (1 mg/ml apotransferrin, 20 mM Putrescine, 4 μM progesterone, and 6 μM selenium; formulation based on Bottenstein and Sato, 197940), 50 nM hydrocortisone, and 0.1 μg/ml insulin (all reagents from Sigma) ] were added. Cultures were fed every 2 days by removing 500 µl of media and replacing it with fresh DM media, after 12 days insulin was removed from the DM used to feed the cultures. The effect of recombinant human FGF9 and other soluble factors (all obtained from R&D Systems) on these in vitro myelinating cultures was investigated by adding them to the culture media from day 18 (i.e., when myelination has just begun) to day 28 in vitro.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from myelinating cultures grown in the presence or absence of FGF9 (100 ng/ml) for 24h and 10 days using the Qiagen RNeasy Micro kit according to the manufacturer's instructions. RNA quality and integrity were checked using the Agilent Bioanalyzer 6000 Nano LabChip platform.
Label
biotin
Label protocol
The total RNAs were processed and labelled with biotin using the Ambion® WT Expression Kit following the Affymetrix GeneChip® WT Terminal Labeling and Hybridization protocol.
Hybridization protocol
The processed RNAs were hybridized to Affymetrix GeneChip® Rat Gene 1.0 ST Arrays using the manufacturer's protocols for using the Fluidics Station 450.
Scan protocol
The hybridized arrays were scanned on the Gene Array Scanner 3000-7G.
Description
CL2RatGe1Control4
Data processing
The data analysis was carried out in Partek Genomics Suite (version 6.5) software. The probeset-level data were normalised using the GCRMA normalisation method and summarised to the transcript cluster level using the One-Step Tukey's Biweight method.
