Project description:Femur and tibia bones were harvested from 8-week-old wild-type (WT) C57BL/6 mice. Bone marrow was flushed out into cold PBS (Life Technologies) plus 2% heat-inactivated FBS, passed through a needle five times to dissociate the cells, and then passed through a 70-μm cell strainer (Becton Dickinson) to remove cell clumps, bone, hair, and other cells/tissues. After addition of three volumes of NH4Cl solution (0.8% NH4Cl solution; Beyotime Institute of Biotechnology, Jiangsu, China), the mixture was incubated on ice for 10 min to remove red blood cells; the cells were then spun down and resuspended in cold PBS with 2% FBS. The harvested cells were cultured in DMEM containing 10% FBS and supplemented with 10 ng/ml recombinant mouse CSF1 (R&D Systems) or 10 ng/ml recombinant mouse CSF2 (R&D Systems) for 7 days to obtain CSF1- or CSF2-induced BMDMs: M(CSF1) or M(CSF2) cells, respectively. To test the difference genes expression of TAMs obtained from M(CSF1) and M(CSF2) cells in C57BL/6 mice. We used microarrays to detail the global programme of gene expression and identified M(CSF1) and M(CSF2) cells during this process.
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:Tissue macrophages from peritoneal cavity, lung, liver, spleen, small intestine and adipose tissue and M-CSF derived bone marrow derived macrophages (BMDMs) were determined for gene expression. Macrophages from six different tissues and BMDMs were compared for gene expression.
Project description:Bone marrow-derived macrophages (BMDMs) are a key model system for studying macrophage biology in vitro. Commonly used methods to differentiate macrophages from bone marrow are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is known about the composition of L929 conditioned media (CM) and how it affects BMDM phenotype. Here, we used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a two-week period. While M-CSF is very abundant in L929 CM, we identified several other immune-regulatory proteins at surprisingly high abundance. L929 CM induced a slightly pre-activated phenotype and the expression of a number of known innate immune proteins in macrophages. This resource will be valuable to all researchers using L929 CM for the differentiation of BMDMs.
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.