Purified astrocyte cultures were exposed to four different experimental stimulation conditions. (i) Basal; consisting of 32hr in stimulation medium (L-glutamine-free DMEM supplemented with 5% CS, 2mM L-glutamine, 50 IU/ml penicillin, and 50μg/ml streptomycin). (ii) TGF-β1; consisting of 32hr in stimulation medium with TGF-β1 (3ng/ml; human recombinant; R&D Systems) (iii) LPS+IFNγ; consisting of 24hr in stimulation medium prior to addition of 2µg/ml LPS (E.coli 0127:B8, Sigma) and 3ng/ml IFNγ (recombinant mouse, R&D Systems) for a further 8hr. (iv) TGFβ+LPS+IFNγ; consisting of 24hr in stimulation medium with TGF-β1 prior to addition of LPS+IFNγ for a further 8hr. The 24hr duration of TGF-β1 treatment was chosen on the basis of previous experiments showing a maximal and stable effect of TGF-β on induction of iNOS and enhancement of nitric oxide production in purified astrocyte cultures over this time period (Hamby et al., 2006a).
Growth protocol
Primary astrocyte cultures were prepared from cerebral cortices of postnatal (1-3 days) C57Bl/6 mice as previously described (Hamby et al., 2006a; Hamby et al., 2006b). In brief, plating media consisted of L-glutamine-free DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone), 10% calf serum (CS, Hyclone), 2mM L-glutamine, 50 IU/ml penicillin, 50µg/ml streptomycin, and 10ng/ml epidermal growth factor (EGF). Upon confluence, astrocyte cultures were purified of microglia by treatment with 8µM cytosine β-D-arabinofuranoside (Ara-C; Sigma) for 5-6 days to substantially reduce microglia followed by treatment with 75mM L-leucine methyl ester (LME, Sigma; 60-90min) one day prior to experimentation to completely eradicate any residual microglia. Cells were maintained in medium consisting of L-glutamine-free DMEM, 10% CS, 2mM L-glutamine, 50 IU/ml penicillin and 50µg/ml streptomycin. All cultures were maintained at 37°C in a humidified atmosphere of 6% CO2 and used after 14-31 days in vitro.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from astrocytes using the Qiagen RNeasy Kit. RNA quantity and quality was assessed using a Nanodrop (Nanodrop Technologies) and the Agilent Bioanalyzer (Agilent Technologies), respectively. Total RNA (200ng) was amplified, biotinylated and hybridized on Illumina Mouseref-8 v2.0 BeadChip arrays. Slides were scanned using Illumina BeadStation and signal extracted using the Illumina BeadStudio software (Illumina, San Diego CA).
