Project description:We derived B-lineage cells by in vitro culture of neonatal cord blood CD34+ cells on MS-5 stromal cells with recombinant IL-7. These cultures yielded CD19+CD127+ and CD19+CD127- cell populations. We performed gene expression profiling on these populations and compared these with each other and with published expression profiles of freshly isolated BM precursor B-cell subsets (E-MEXP-384). These analyses yielded new insights into their different functionality and developmental stage.<br><br>A file containing statistical analysis of the normalized data is included in the file named E-MEXP-2878.additional.zip on the FTP site for this experiment.
Project description:Synchronized sexuparae were randomly divided into 2 batches and treated with kinoprene or acetone (as a control) 24h after fourth instar moult (Fig. S1). 25 treated sexuparae were collected 24, 48, and 72 hours after kinoprene or acetone application. At these 3 times the most developed embryos respectively correspond to the developemental stage 18, 19 or 20. For each condition, the 5 most developed embryos were isolated from each of the 25 treated sexuparae by dissection, pooled together, frozen into liquid nitrogen and stored at -80 C. This procedure was repeated 5 times to generate as much independent biological replicates. Total RNAs were isolated from each sample by using the RNeasy Mini kit (Qiagen) according to manufacturer's instructions. RNA quality was checked on Bioanalyser (Agilent) and quantified on Nanodrop (Thermo scientific). For each sample 20µg of total RNAs were sent to the NimbleGen expression array platform (Roche). Double stranded cDNA synthesis and Cy3 end-labelling were performed by NimbleGen.
Project description:Fibroblasts, Serum-free Fibrocytes, Serum-containing Fibrocytes, Monocytes, Macrophages, Osteoclasts, immature Dendritic Cells, and mature Dendritic Cells were all generated from 3 biological replicates from each of 3 separate donors. RNA was extracted (Ambion RNAqueous), labelled with cy3, mixed with cy5 labelled human reference (Stratagene), and hybridised to slides printed with Human AROS v4.0 oligonucleotides (Operon). Slides were scanned using a Perkin Elmer GX plus, and the data then normalised with GEPAS v4.0 and collated. Final data analysis was carried out using TMEV 4.0. SAM was performed using a 0.1% FDR. HCL and PCA were plotted from this list, and interrogation carried out using DAVID to determine pathway enrichment. After initial analysis, the Fibroblast cell type was found to significantly bias the data. Due to this, these samples were removed from the analysis, and the data re-normalised. This second data file will be submitted separately.
Project description:At one site (#10), three different batches of MTRRM (see E-TABM-16), were labeled with two different kits (Enzo and Affymetrix) and hybridized to two different Affymetrix Arrays (RAE230A and RAE230_2).
Project description:Generation of monocyte-derived dendritic cells (DC). Immature DC were prepared from peripheral blood mononuclear cells (PBMCs) isolated from whole blood of healthy donors. A minimum of 3 individual donor blood was used for each experiment. PBMCs were incubated with anti-CD14 microbeads (Miltenyi Biotec, Bergish Gladbach, Germany) as specified in the manufacturer's instructions. The isolated monocytes were resuspended at 10^6 cells/ml in medium supplemented with 10% human AB serum, 100 ng/ml GM-CSF (Leucomax; Novartis, Camberley, UK) and 50 ng/ml IL-4 (Peprotech EC Ltd, London, UK), cultured in tissue culture flasks. On day 7 the cells were defined as immature DC with low/intermediate HLA-DR expression. Microarray analysis of the effects of Paclitaxel and LPS on DC. Total RNA was extracted from day 7 DC which had been treated for 2 h with 1 ug/ml LPS or 100 uM paclitaxel for 2 h then washed and returned to culture for 24 h. This was used as a template to generate Cy3-labelled cRNA, using the Low RNA Input Linear Amplification Kit (Agilent). This was used as a probe on the Whole Human Genome Microarray (4x44K) slide (Agilent). Slides were scanned using the Agilent scanner and data extracted using Feature Extraction Software 9.5.3 (Agilent). Subsequent data analysis was performed using GeneSpring GX software.
Project description:Potato genotypes from a diploid potato population were divided in two groups based on their response to Potato virus A (PVA). Plants exhibiting hypersensitive response were compared to plants exhibiting non-necrotic response (i.e. blocking virus movement without cell death).<br>The comparisons were made before inoculation and 12 and 24 hours post-inoculation.<br>
Project description:Toll-like receptor (TLR) signalling activation by pathogens is critical to the induction of immune responses, and demands tight regulation. Chemokine ligand 2 (CCL2) secretion triggered by TLR4 or TLR8 engagement is strongly inhibited upon simultaneous activation of both TLRs in human monocyte-derived dendritic cells (MD-DC). Impaired CCL2 secretion occurs concomitantly to IL-12 up-regulation, being part of a complex regulatory circuit ensuring optimal Th type 1 polarization. Interestingly, triggering selected TLRs or their combinations differently affects nuclear factor-kB p65 activation and microRNA expression. To investigate in details such different modulation we performed a microarray profiling of MD-DCs stimulated by different TLRs agonist or their combination in three different donors. We found that CCL2 supplies an important immunomodulatory role to DCs, and may contribute to dictate the cytokine profile in Th type 1 responses induced by DCs.