6 protocols
AccessionType
array scanning protocol
750ng of labelled cRNA was hybridised to a Rat Ref-12 Beadchip & scanned by the BeadArray Reader
nucleic acid hybridization to array protocol
750ng of labelled cRNA was hybridised to a Rat Ref-12 Beadchip & scanned by the BeadArray Reader
nucleic acid labeling protocol
100ng and were processed according to the Illumina Whole-Genome Gene Expression Direct Hybridization Assay Guide, using the Ambion Kit: Illumina TotalPrepÈ-96 RNA Amplification Kit (LSN-X-036). Qualitative & quantitative Qcs were performed on the labelled cRNA (LSN-X-013 & LSN-X-024) and 750ng of labelled cRNA was hybridised to a Rat Ref-12 Beadchip & scanned by the BeadArray Reader (LSN-X-036).
treatment protocol
1 micromole dexamethasone was applied to cells for 24 hours.
nucleic acid extraction protocol
RNeasy Mini Kit (Qiagen, UK)
growth protocol
Cell cultures: Primary mixed glial cultures were prepared from dissociated cerebral cortices of Sprague-Dawley rats at postnatal day 1 _ 3 and glial populations were isolated by sequential rotary shaking procedures using well-established protocol. Microglia were derived first (200 rpm, 1 h) and plated at 6 x 104 cells/cm2 in D10 medium [Dulbeccoês modified Eagleês medium (DMEM) supplemented with 10percent fetal bovine serum (FBS), 2 mM glutaMAX-I, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 microgram/ml streptomycin]. OPCs were derived next (200 rpm, 18 h) and plated at 3 x 104 cells/cm2 in OPC maintenance medium (OPC-MM: DMEM supplemented with 2 mM glutaMAX-I, 1 mM sodium pyruvate, 10 nM biotin, 10 nM hydrocortisone, 30 nM sodium selenite, 50 microgram/ml transferrin, 5 microgram/ml insulin, 0.1percent bovine serum albumin, 50 U/ml penicillin, 50 microgram/ml streptomycin, 10 ng/ml PDGF-AA, and 10 ng/ml FGF2). Finally, after an additional shake (200 rpm, 18 h) to deplete residual OPCs, adherent astrocytes were trypsinised and plated at 4 x 104 cells/cm2 in D10. To deplete microglia from OPC and astrocyte fractions, cells were transferred to non-tissue-culture grade petri dishes (to which microglia readily attach) and after 30 min, the unattached cells were collected. To establish oligodendrocyte cultures, OPC cultures were switched to Sato differentiation medium [DMEM supplemented with 2 mM glutaMAX-I, 1 mM sodium pyruvate, 1x N2 supplement (5 microgram/ml insulin; 20 nM progesterone; 100 µM putrescine; 30 nM selenium; 100 microgram/ml transferrin), 30 nM thyroxine, 30 nM triiodothyronine, 50 U/ml penicillin, and 50 microgram/ml streptomycin]. All cultures were incubated at 37degrees C in 5percent CO2/95percent humidified air.