The in vitro myelinating cultures were either untreated or treated with FGF1, FGF2, FGF9 or BMP4. 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 confluent monolayer. 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 FGFs or BMP4 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 using the Qiagen Rneasy Micro kit according to manufacturer's instructions
Label
biotin
Label protocol
labelled with biotin using Ambion WT Expression Kit following the Affymetrix GeneChip WT Terminal Labeling and Hybridization protocol
Hybridization protocol
hybridized to Affymetrix Rat Gene 1.0 ST Arrays using manufacturer's protocols using Fluidics Station 450
Scan protocol
Gene Array Scanner 3000-7G
Description
gene expression data from myelinating cultures 28 DIV treated with BMP4
Data processing
Partek Genomics Suite (version 6.6), probeset level data were normalized using GCRMA normalisation and summarised to transcript cluster level using One-Step Tukey's Biweight method.
