primary mouse astrocytes cultures were prepared from newborn c57bl/6jolahsd mice brains as previously described . after removing meninges and large blood vessels , brains cells were cultivated in dulbecco 's modified eagle medium supplemented with 10% fetal bovine serum , 100  u / ml penicillin and 100  g / ml streptomycin at 37  c in a humidified atmosphere containing 5% co2 . the culture medium was changed after three days , and cultures reached confluence after 1014  days . then , glial cells were separated by a magnetic cell sorting ( macs ) method according to the manufacturer 's protocol ( miltenyi biotec , the netherlands ) . briefly , glial cultures were trypsinized and microglia , the cd11b - positive cells present in the astrocyte monolayer , were collected by a positive selection . astrocyte - enriched cultures were obtained by platting the cells in 75  cm flasks . after 3  days , the culture medium was replaced and after 7  days , when cultures reached confluence , the macs procedure was repeated in order to reduce the residual microglial contamination in our astrocyte population . after additional 7  days , cultures of primary mouse astrocytes were treated with tnf ( 50  ng / ml ; r&d systems , united kingdom ) during 24  h. total rna was extracted using rna now reagent ( ozyme , france ) according to the manufacturer 's instructions . to determine the effects of tnf on primary astrocytes , all samples were of high purity and integrity and were assessed by the agilent 2100 bioanalyzer and rna 6000 nano labchip kits ( agilent technologies ) . microarray gene expression data was normalized using the gc - rma procedure with default parameters for background correction , quantile normalization , and probe replicate summarization . differentially expressed genes between control and tnf conditions were then determined using the empirical bayes moderated t - statistic ( ebayes ) . p - value significance scores for these genes were adjusted for multiple hypotheses testing according to the benjamini a heat map and dendrogram cluster visualization for the top 150 most significant known genes ( fig . 1 ) was obtained using standard hierarchical average linkage clustering with a euclidean distance metric . a volcano plot for the analysis of differential gene expression between tnf and control samples the negative decadic logarithm of the adjusted p - value significance score was plotted against the logarithm of the fold change . several genes ( green dots ) are significantly altered ( adjusted p  <  0.05 ) and display an absolute log fold change above 1 in expression ( fig . alterations in known cellular pathways and processes were identified and visualized by applying the metacoretm genego software onto the differential expression statistics obtained from the ebayes analysis . the genes were pre - filtered using a significance threshold ( adjusted p - value <  0.05 ) before applying the default genego pathway analysis . pathway analysis with genego revealed that pathways related to glial differentiation , immune response and apoptosis were modulated ( fig . primary mouse astrocytes cultures were prepared from newborn c57bl/6jolahsd mice brains as previously described . after removing meninges and large blood vessels , brains cells were cultivated in dulbecco 's modified eagle medium supplemented with 10% fetal bovine serum , 100  u / ml penicillin and 100  g / ml streptomycin at 37  c in a humidified atmosphere containing 5% co2 . the culture medium was changed after three days , and cultures reached confluence after 1014  days . then , glial cells were separated by a magnetic cell sorting ( macs ) method according to the manufacturer 's protocol ( miltenyi biotec , the netherlands ) . briefly , glial cultures were trypsinized and microglia , the cd11b - positive cells present in the astrocyte monolayer , were collected by a positive selection . astrocyte - enriched cultures were obtained by platting the cells in 75  cm flasks . after 3  days , the culture medium was replaced and after 7  days , when cultures reached confluence , the macs procedure was repeated in order to reduce the residual microglial contamination in our astrocyte population . after additional 7  days , cultures of primary mouse astrocytes were treated with tnf ( 50  ng / ml ; r&d systems , united kingdom ) during 24  h. total rna was extracted using rna now reagent ( ozyme , france ) according to the manufacturer 's instructions . to determine the effects of tnf on primary astrocytes , mrna samples were analyzed by affymetrix genechip mouse gene 1.0 st arrays . all samples were of high purity and integrity and were assessed by the agilent 2100 bioanalyzer and rna 6000 nano labchip kits ( agilent technologies ) . microarray gene expression data was normalized using the gc - rma procedure with default parameters for background correction , quantile normalization , and probe replicate summarization . differentially expressed genes between control and tnf conditions were then determined using the empirical bayes moderated t - statistic ( ebayes ) . p - value significance scores for these genes were adjusted for multiple hypotheses testing according to the benjamini hochberg procedure . a heat map and dendrogram cluster visualization for the top 150 most significant known genes ( fig . 1 ) was obtained using standard hierarchical average linkage clustering with a euclidean distance metric . a volcano plot for the analysis of differential gene expression between tnf and control samples the negative decadic logarithm of the adjusted p - value significance score was plotted against the logarithm of the fold change . several genes ( green dots ) are significantly altered ( adjusted p  <  0.05 ) and display an absolute log fold change above 1 in expression ( fig . alterations in known cellular pathways and processes were identified and visualized by applying the metacoretm genego software onto the differential expression statistics obtained from the ebayes analysis . the genes were pre - filtered using a significance threshold ( adjusted p - value <  0.05 ) before applying the default genego pathway analysis . pathway analysis with genego revealed that pathways related to glial differentiation , immune response and apoptosis were modulated ( fig . . these expression data could be useful to describe the effect of the nfb activation on primary astrocyte cultures devoid of microglia . taking advantage of the macs technology , in contrast to the main studies reported in the literature , we were able to characterize pure populations of astrocytes under inflammatory conditions . we show that tnf increases the expression of genes associated with the nfb pathway and induces the re - expression of genes implicated in glial developmental processes . these data highlight the importance of the nfb pathway during the conversion of astrocytes into reactive cells and , particularly , its active role in the dedifferentiation process .