Project description:In vitro experiments: Proteomic profiling of bone marrow-derived macrophages following 24-hour stimulation with LPS, IL-4, and TGF-β. In vivo experiments: Proteomic analysis of spinal cord changes following transplantation of bone marrow-derived macrophages induced by LPS, IL-4, and TGF-β in a spinal cord injury model.
Project description:Bone marrow-derived macrophages, Unstimulated DMSO Bone marrow-derived macrophages, Unstimulated + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 1h DMSO Bone marrow-derived macrophages, LPS 1h + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 2h DMSO Bone marrow-derived macrophages, LPS 2h + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 4h DMSO Bone marrow-derived macrophages, LPS 4h + I-BET (GSK525762A) LPS (100 ng/mL) was purchased from Sigma. Bone Marrow-derived macrophages (BMDMs) were differentiated from C57BL/6 bone marrow using 5 ng/mL each of recombinant M-CSF and IL-3 (Peprotech) for 7 days as described (Jeffrey et al, Nature Immunology, 2006). 2 x10^6 BMDMs were treated with DMSO or 1 μM of I-BET for 30 minutes before the addition of LPS (100 ng/mL) for 1, 2 or 4h. Unstimulated control samples were incubated with I-BET only for 1 hour. 500 ng of total RNA from 3 independent samples per group was used to prepare biotin-labeled RNA using Ambion Illumina TotalPrep RNA Amplification Kit (Applied Biosystems) and hybridized to Illumina MouseRef-8 v2.0 expression BeadChip kits. The chips were scanned using Illumina BeadArray Reader.
Project description:Untargeted metabolites of supernatant of wild-type and Nlrp3-/- bone marrow-derived macrophages without stimulation or with 4 hours of LPS preconditioning and subsequently 30 min of ATP stimulation.
Project description:Bone marrow-derived macrophages, Unstimulated DMSO Bone marrow-derived macrophages, Unstimulated + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 1h DMSO Bone marrow-derived macrophages, LPS 1h + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 2h DMSO Bone marrow-derived macrophages, LPS 2h + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 4h DMSO Bone marrow-derived macrophages, LPS 4h + I-BET (GSK525762A) LPS (100 ng/mL) was purchased from Sigma. Bone Marrow-derived macrophages (BMDMs) were differentiated from C57BL/6 bone marrow using 5 ng/mL each of recombinant M-CSF and IL-3 (Peprotech) for 7 days as described (Jeffrey et al, Nature Immunology, 2006). 2 x10^6 BMDMs were treated with DMSO or 1 μM of I-BET for 30 minutes before the addition of LPS (100 ng/mL) for 1, 2 or 4h. Unstimulated control samples were incubated with I-BET only for 1 hour. 500 ng of total RNA from 3 independent samples per group was used to prepare biotin-labeled RNA using Ambion Illumina TotalPrep RNA Amplification Kit (Applied Biosystems) and hybridized to Illumina MouseRef-8 v2.0 expression BeadChip kits. The chips were scanned using Illumina BeadArray Reader. 3 biological replicates and 4 timepoints
Project description:The activation profiles of macrophages under different immune and inflammatory conditions have generated great interest. LPS, in particular, is a commonly used in vitro model of infection and inflammation studies in macrophages. We have used gene expression microarrays to define the effects of each of three variables; LPS dose, LPS vs. interferons beta and gamma, and genetic background on the transcriptional response of mouse bone marrow-derived macrophages Macrophages derived from the C57BL/6 strain of mouse were challenged with increasing doses of LPS over a 24 hour time-course (0.5ng/ml, 5ng/ml, or 50ng/ml LPS). BALB/c derived macrophages were treated with any of 5ng/ml LPS, 10U/ml of recombinant mouse interferon-beta, or 10U/ml interferon-gamma, over a 24 hour timecourse. Sampling times were 0 hours (pre-treatment) and 1, 2, 4, 8, and 24 hours post-treatment in each case.
Project description:Innate immune response to pathogens is a complex, multi-staged process involving thousands of genes. While numerous transcription factors that act as master regulators of the response to LPS have been identified, the temporal complexity of gene expression changes strongly suggest that additional layers of regulation remain to be uncovered. This study was done to examine response of mouse bone marrow-derived macrophages to stimulation with LPS over time. Bone marrow-derived macrophages from C57BL/6J mice were incubated in the presence of LPS for 1, 4 or 12 h, while the control group was incubated under same conditions for 4 h in the absence of the LPS. RNA isolated from these macrophages was analyzed using the Affymetrix Mouse Exon Array 1.0 ST.
Project description:Gene expression in bone marrow-derived macrophages (BMDMs) from WT and mice lacking the transcriptional repressor Kruppel-like factor 3 (KLF3). We cultured BMDMs from bone marrow for 7-10 days then treated cells with 100 ng/mL lipopolysaccharide (LPS) or vehicle (PBS) for 0 h or 8 h, followed by RNA extraction. We aimed to investigate deregulated genes and pathways in macrophages lacking KLF3, during the inflammatory response to endotoxin (LPS).
Project description:The activation profiles of macrophages under different immune and inflammatory conditions have generated great interest. LPS, in particular, is a commonly used in vitro model of infection and inflammation studies in macrophages. We have used gene expression microarrays to define the effects of each of three variables; LPS dose, LPS vs. interferons beta and gamma, and genetic background on the transcriptional response of mouse bone marrow-derived macrophages Macrophages derived from the C57BL/6 strain of mouse were challenged with increasing doses of LPS over a 24 hour time-course (0.5ng/ml, 5ng/ml, or 50ng/ml LPS). BALB/c derived macrophages were treated with any of 5ng/ml LPS, 10U/ml of recombinant mouse interferon-beta, or 10U/ml interferon-gamma, over a 24 hour timecourse. Sampling times were 0 hours (pre-treatment) and 1, 2, 4, 8, and 24 hours post-treatment in each case. 64 samples in total were analysed. These incorporated 6 different timecourse studies. A combination of statistical filtering using the Empirical Bayes function in Bioconductor package (R statistical software), and co-expression analysis using the network analysis tool BioLayout Express 3D, was used to compare the timecourse studies.
Project description:We cultured bone marrow derived dendritic cells from WT and CD11c KO mice. Then, a group of bone marrow dendritic cells were stimulated with LPS overnight. We obtained bone marrow derived dendritic cells with or without LPS stimulation and analyzed proteomics profiles.
Project description:Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-loaded macrophages in the arterial wall. Intimal macrophages internalize modified lipoproteins such as oxidized LDL (oxLDL) through scavenger receptors, leading to storage of excess cholesteryl esters in lipid bodies and a "foam cell" phenotype. In addition, stimulation of macrophage Toll-like receptors (TLRs) has been shown to promote lipid body proliferation. We investigated the possibility that there are transcriptional regulators that are common to both pathways for stimulating foam cell formation (modified lipoproteins and TLR stimulation), and identified the transcription factor ATF3 as a candidate regulator. In this specific microarray experiment, we studied the transcriptional response of murine macrophages to stimulation with the TLR4 agonist, LPS. Bone marrow-derived macrophages from C57BL/6J mice were incubated in the presence or absence of LPS for 4 h, and then transcriptionally profiled using the Affymetrix Mouse Exon Array 1.0 ST. The goal was to study the pattern of transcript-level differential expression between the unstimulated and LPS-stimulated cells. Three female mice (strain C57BL/6J) were sacrificed at 8-12 weeks of age, and macrophages were derived from the femoral bone marrow using rhM-CSF. On day six, macrophages were divided into two treatment groups per animal, for a total of six samples. LPS was introduced into the medium for three samples (one from each animal) at 10 ng/mL on day seven, and after 4 h of incubation, RNA was isolated using Trizol. Labeled cRNA derived from the RNA samples was hybridized to Affymetrix Mouse Exon Array 1.0 ST GeneChips. Microarray data were processed using transcript-level probesets, and are in log2 scale.