Project description:We evaluated both the transcriptomic and inflammatory response in trout (O. mykiss) macrophages in primary cell culture stimulated with DAP-PGN (DAP; meso-diaminopimelic acid, PGN; peptidoglycan) from two strains of Escherichia coli (PGN-K12 and PGN-O111:B4) over time. Transcript profiling was assessed using function-targeted cDNA microarrays hibridation (n = 36) to differential responses to both PGNs that are both time and treatment dependen over trout macrophages. Wild type E. coli (K12) generated an increase in transcript number/diversity over time whereas PGN-O111:B4 stimulation resulted in a more specific and intense response. In line with this gene Ontology analysis (GO) highlights a specific transcriptomic remodelling for PGN-O111:B4 whereas results obtained for PGN-K12 shows a high similarity with a general LPS response where multiple functional classes are related to ribosome biogenesis or cellular metabolism Two-condition experiment, PGN vs. control cells. Biological replicates: 18 control, 18 treated. Dye-swap.
Project description:We evaluated both the transcriptomic and inflammatory response in trout (O. mykiss) macrophages in primary cell culture stimulated with DAP-PGN (DAP; meso-diaminopimelic acid, PGN; peptidoglycan) from two strains of Escherichia coli (PGN-K12 and PGN-O111:B4) over time. Transcript profiling was assessed using function-targeted cDNA microarrays hibridation (n = 36) to differential responses to both PGNs that are both time and treatment dependen over trout macrophages. Wild type E. coli (K12) generated an increase in transcript number/diversity over time whereas PGN-O111:B4 stimulation resulted in a more specific and intense response. In line with this gene Ontology analysis (GO) highlights a specific transcriptomic remodelling for PGN-O111:B4 whereas results obtained for PGN-K12 shows a high similarity with a general LPS response where multiple functional classes are related to ribosome biogenesis or cellular metabolism
Project description:The goal of this experiment is to profile LPS-stimulated transcriptome changes in human macrophages. Human whole blood was from the Sanofi in-house blood donor service that is approved by the local ethics committee and all blood donors signed informed consent. Human peripheral blood monocytes were isolated from anonymous donors’ prefinal blood using Ficoll density centrifugation, followed by magnetic separation with positive selection (CD14 MicroBeads, Miltenyi Biotec). Monocytes were differentiated into macrophages by culturing in macrophage serum-free medium (Life Technologies) containing 50 ng/ml recombinant human macrophage colony-stimulating factor (Immunotools) for 5 days. Following differentiation, cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (Thermo Fisher) and maintained at 37°C in a 5% CO2/air environment. Macrophages were stimulated with 50 ng/ml lipopolysaccharides from Escherichia coli O111:B4 (Sigma) for 24 hours.
Project description:Murine splenocytes were isolated from the spleens of C57BL/6J mice and were pretreated in vitro for three days in the presence of Pam3CSK4 (1 μg/ml), high pure LPS from E.coli O111:B4 (100 ng/ml) and R848 (5 μg/ml). PBS-treated splenocytes served as negative control. We used Qiagen Toll-like Receptor RT2 Profiler PCR Array kit to quantitate gene expression profiling of the TLR signaling pathway.
Project description:Transcriptional profiling of BV-2 microglial cells comparing control untreated BV-2 cells with LPS-treated BV-2 cells or obovatol/LPS-treated BV-2 cells. Objective was to determine the effect of obovatol on LPS-induced gene expression in microglia.
Project description:We have employed whole genome microarray expression profiling to identify differently expressed genes following stimulation of wild-type bone-marrow derived macrophages with Escherichia coli lipopolysaccharide (LPS). Macrophages were stimulated with LPS (100 ng/ml) for 6 hours and a signature was identified that distinguished between infected and control samples. Expression of several genes from this signature was quantified in the same RNA samples by real-time PCR, confirming the predicted macrophage response pattern.
Project description:We have employed whole genome microarray expression profiling to identify differently expressed genes following stimulation of interleukin-16-knock-out bone-marrow derived macrophages with Escherichia coli lipopolysaccharide (LPS). Macrophages were stimulated with LPS (100 ng/ml) for 6 hours and a signature was identified that distinguished between infected and control samples. Expression of several genes from this signature was quantified in the same RNA samples by real-time PCR, confirming the predicted macrophage response pattern.
Project description:This study describes the development and validation of the Aquagenomic Sparus aurata oligonucleotide-microarray (SAQ) based on the Agilent Technology system (eArray) to provide a platform for studies on the gene expression of gilthead seabream. The platform developed used all available public ESTs stored and annotated in the Aquagenomic Consortium seabream library (10K). In fish, lipopolysaccharide (LPS) gives a robust cytokine response that is stimulated by crude LPS preparations, some component of the LPS complex are responsible for this stimulation. Peptidoglycan (PGN) is a component of G-negative bacteria (found as a contaminant of crude LPS preparations) able to be recognized by macrophages inducing depth transcriptional modulations and a strong inflammatory response. For microarray analysis, head kidney macrophage cultures were used (N = 36 fish). Each cell culture was stimulated with equal concentrations of PGN and LPS from E. coli O111:B4 strain (10 ug/mL): non-stimulated cell cultures (control n = 9 fish), stimulated during 6 h with LPS (n = 9), and stimulated during 1 h (n = 9), and 6 h (n = 9) with PGN. A loop microarray design approach was used for the study. All experimental RNA samples were labelled with a single colour dye (Cy3) and each stimulated sample was compared to the control sample (pool without stimulation) labelled with the same dye (Cy3). Our microarray analyses identified differential transcriptional modulations in macrophages stimulated with both LPS and PGN at the level of differentially activated RNA transcripts related with the regulation of transcriptional program, prostaglandin synthesis or highlighting the expression of responsive gene-cassettes tightly related to LPS-PGN host recognition.
Project description:Murine splenocytes were isolated from the spleens of C57BL/6J mice and were pretreated in vitro for three days in the presence of PBS, Pam3CSK4 (1 μg/ml), high pure LPS from E.coli O111:B4 (100 ng/ml) and R848 (5 μg/ml). BALB/c irradiated recipients, were transplanted with T-cell depleted bone marrow alone, or with the aforementioned pretreated splenocytes, and 10 days after, PBMCs from all experimental groups were collected for TLR signaling pathway analysis.We used Qiagen Toll-like Receptor RT2 Profiler PCR Array kit to quantitate gene expression profiling of the TLR signaling pathway.
Project description:We have employed whole genome microarray expression profiling to identify differently expressed genes following stimulation of interleukin-16-knock-out bone-marrow derived macrophages with Escherichia coli lipopolysaccharide (LPS). Macrophages were stimulated with LPS (100 ng/ml) for 6 hours and a signature was identified that distinguished between infected and control samples. Expression of several genes from this signature was quantified in the same RNA samples by real-time PCR, confirming the predicted macrophage response pattern. Gene expression in macrophages was measured at 6 hours after stimulation Three independent experiments were performed.