аЯрЁБс > ўџ 5 7 ўџџџ 4 џџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџьЅС a јП ф jbjbtt " э э ф џџ џџ џџ Ш Ш Ш Ш Ш Ш Ш 2 њ њ њ њ 2 x p i i i §
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Gene array analysis. Microglia were cultured on PDL or CSPG for 36 h, and their RNA was purified as described above ADDIN EN.CITE Butovsky200650162976373112006JanMicroglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells149-60Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel.Butovsky, O.Ziv, Y.Schwartz, A.Landa, G.Talpalar, A. E.Pluchino, S.Martino, G.Schwartz, M.Mol Cell Neuroscihttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=162976371. Array processing was performed using Affymetrix MOE430A oligonucleotide arrays. Total RNA from each sample was used to prepare biotinylated target RNA, with minor modifications according to the manufacturer's recommendations. Briefly, 5 Еg of mRNA was used to generate first-strand cDNA by using a T7-linked oligo(dT) primer. After second-strand synthesis, in-vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics, Farmingdale, NY), resulting in an approximately 300-fold amplification of RNA. The target cDNA generated from each sample was processed as recommended by the manufacturer, using an Affymetrix GeneChip. Instrument System. Briefly, spike controls were added to 15 Еg fragmented cRNA and hybridized ovenight. Arrays were then washed and stained with streptavidin-phycoerythrin before being scanned on an Affymetrix GeneChip scanner. The qualities and amounts of starting RNA wer e c o n f i r m e d u s i n g a n a g a r o s e g e l . A f t e r s c a n n i n g , g e n e a r r a y i m a g e s w e r e a s s e s s e d b y e y e t o c o n f i r m s c a n n e r a l i g n m e n t a n d t h e a b s e n c e o f s i g n i f i c a n t b u b b l e s o r s c r a t c h e s o n t h e c h i p s u r f a c e . 3 ' / 5 ' r a t i o s f o r G A P D H a n d b№- a c t i n w e r e v e r i f i e d t o b e w i t h i n a c ceptable limits (1.03-1.14 and 0.78-0.86), and BioB spike controls were present on all chips, with BioC, BioD, and CreX also present at increasing intensities. When scaled to a target intensity of 150 (using Affymetrix MAS 5.0 gene array analysis software), scaling factors for all arrays were within acceptable limits (0.63-0.84), as were background, Q values, and mean intensities. Details of quality control measures can be found at ADDIN EN.CITE http://apps1.niaid.nih.gov/David/upload.asp30http://apps1.niaid.nih.gov/David/upload.asp2.
Data analysis. The probe sets contained in the Affymetrix MOE430A oligonucleotide arrays were filtered using a Mas 5 algorithm. A list of "valid" probe sets was obtained, representing probe sets with signals higher than 20 and found to be present (P) in at least one sample. A comparison of treated and control samples yielded a list of "active genes" (representing probe sets changed by at least 2 fold, calculated from the MAS 5 log ratio values, LR ( 1 or LR ( 1) and detected as "Increased" or as "Decreased" in at least one time point. Excluded from this list were up-regulated genes in all treated samples with signals lower than 20 or recorded as absent, and down-regulated genes with baseline signals lower than 20 and recorded as absent in the control samples. Genes were classified into functional groups using the GO annotation tool (see ADDIN EN.CITE http://eng.sheba.co.il/genomics40http://www.ncbi.nlm.nih.gov/geo/ or at http://eng.sheba.co.il/genomics3). Selected genes from each group are presented.
Flow cytometric analysis. Mice were subjected to spinal cord injury; after 7 days they were killed and their bone-marrow cells were harvested for FACS analysis. Bone marrow cells were isolated as described in the main text. Fluorochrome-labeled mAbs were purchased from BD Pharmingen or eBioscience and used according to the manufacturers protocols. Cells were analyzed on a FACSCalibur cytometer (BD Biosciences) using CellQuest software (BD Biosciences).
REFERENCES
ADDIN EN.REFLIST 1. Butovsky, O. et al. Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells. Mol Cell Neurosci 31, 149-60 (2006).
2. HYPERLINK "http://apps1.niaid.nih.gov/David/upload.asp" http://apps1.niaid.nih.gov/David/upload.asp.
3. HYPERLINK "http://eng.sheba.co.il/genomics" http://eng.sheba.co.il/genomics, h.w.n.n.n.g.g.o.a.
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