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 .
 astrocytes , the most abundant glial cell population in the central nervous system , have important functional roles in the brain as blood brain barrier maintenance , synaptic transmission or intercellular communications [ 1 ] , [ 2 ] . numerous studies suggested that astrocytes exhibit a functional and morphological high degree of plasticity . 
 for example , following any brain injury , astrocytes become reactive and hypertrophic . 
 this phenomenon , also called reactive gliosis , is characterized by a set of progressive gene expression and cellular changes [ 3 ] . 
 interestingly , in this context , astrocytes can re - acquire neurogenic properties . 
 it has been shown that astrocytes can undergo dedifferentiation upon injury and inflammation , and may re - acquire the potentiality of neural progenitors [ 4 ] , [ 5 ] , [ 6 ] , [ 7].to assess the effect of inflammation on astrocytes , primary mouse astrocytes were treated with tumor necrosis factor  ( tnf ) , one of the main pro - inflammatory cytokines . the strength of this study is that pure primary astrocytes were used . 
 as microglia are highly reactive immune cells , we used a magnetic cell sorting separation ( macs ) method to further obtain highly pure astrocyte cultures devoid of microglia.here , we provide details of the microarray data , which have been deposited in the gene expression omnibus ( geo ) under the series accession number gse73022 . 
 the analysis and interpretation of these data are included in gabel et al . 
 ( 2015 ) . 
 analysis of gene expression indicated that the nfb pathway - associated genes were induced after a tnf treatment . 
 we have shown that primary astrocytes devoid of microglia can respond to a tnf treatment with the re - expression of genes implicated in the glial cell development . 
Direct link to deposited data
Experimental design, materials and methods
Cell culture and experimental design
Microarrays experiments, quality control and data analysis
Conclusion
Conflict of interest