Project description:Granulocyte-Macrophage colony stimulating factor (GM-CSF) devlops heterogenous myeloid cell populations from bone marrow progenitor cells. In vitro generated bone marrow derived cells are excellent sources for obtaining dendritic cells or macrophages, but it is still not clear about the exact mixed population characteristics of GM-CSF grown cells. We revealed here that GM-CSF grown bone marrow cell derived attaching cells were composed of dendritic cells (GM-BMDC) as well as macrophages (GM-BMM). We compared the transcriptome profiles of these cell populations as well as M-CSF grown bone marrow derived macrophages (M-BMM). We used microarrays to detail the global profile of gene expressions between three populations of CSF-grown bone marrow derived cells: GM-CSF derived dendritic cells (GM-BMDC), GM-CSF derived macrophages (GM-BMM) and M-CSF derived macrophages (M-BMM).
Project description:Granulocyte-Macrophage colony stimulating factor (GM-CSF) devlops heterogenous myeloid cell populations from bone marrow progenitor cells. In vitro generated bone marrow derived cells are excellent sources for obtaining dendritic cells or macrophages, but it is still not clear about the exact mixed population characteristics of GM-CSF grown cells. We revealed here that GM-CSF grown bone marrow cell derived attaching cells were composed of dendritic cells (GM-BMDC) as well as macrophages (GM-BMM). We compared the transcriptome profiles of these cell populations as well as M-CSF grown bone marrow derived macrophages (M-BMM). We used microarrays to detail the global profile of gene expressions between three populations of CSF-grown bone marrow derived cells: GM-CSF derived dendritic cells (GM-BMDC), GM-CSF derived macrophages (GM-BMM) and M-CSF derived macrophages (M-BMM). Bone marrow cells were differentiated for 7 days with 25 ng/ml GM-CSF or 20% L cell conditioned media as a M-CSF supplier. GM-BMDCs were sorted from MHCIIhighF4/80low population and GM-BMMs were sorted in the MHCIIlowF4/80high population. M-BMMs were sorted from CD11b+F4/80+ population.
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:GM-CSF derived bone marrow cultures contain several subsets of CD11c+MHCII+ mononuclear phagocytes Using Affymetrix microarrays we compared gene expression of the different mononuclear phagocytes within the bone marrow culture. The different populations are left unstimulated or are stimulated with LPS
Project description:Mouse bone marrow derived dendritic cells were generated by culturing bone marrow cells at a density of 0.5x10E6 cells/ml in RPMI-1640 supplemented with 5% FCS, 1% Pen/Strep, 5microM 2-mercaptoethanol, 20ng/ml GM-CSF. At day 7 dendritic cells were stimulated or not with 500 ng/ml LPS, and collected at day 10.
2x10E8 cells were used to prepare whole cell extracts and to perform PU.1 immunoprecipitaion with PU.1 antibody (T-21 Santa Cruz). IgG was used as control.
Project description:Bone marrow derived dendritic cells were generated from Ubc9[fl;-] and Ubc9[+/+] mice. After in vitro derivation in the presence of GM-CSF, dendritic cells were treated with tamoxifen for four days to cause CreERT2 activation, and induce Ubc9 floxed allele deletion. This allowed comparative transcriptomic analysis of Ubc9[+/+] and Ubc9[-/-] dendritic cells unstimulated or stimulated with 10ng/ml LPS for one hour and six hours. Three biological replicates of each conditions were used. Analyzed bone marrow derived dendritic cells were Ubc9 WT or Ubc9 KO. Cells were left unstimulated or stimulated with LPS 10 ng/ml for one hour and six hours.
Project description:Purpose: The goals of this study are to compare NGS-derived GM-CSF-cultured bone marrow derived cells transcriptome profiling (RNA-seq) normalized counts and differential expression of genes between different stimulations (untreated, Beauvericin, LPS or Beauvericin with LPS). Methods: mRNA profiles of GM-CSF-cultured bone marrow derived FACS purified MHCII high CD11c+ cells from wild-type C57BL/6N mice that were left untreated or stimulated with Beauvericin, LPS or Beauvericin with LPS for 4h were generated by deep sequencing, in quadruplicate, using the Illumina NextSeq550 system. DNase digested total RNA samples used for transcriptome analyses were quantified (Qubit RNA HS Assay, Thermo Fisher Scientific) and quality measured by capillary electrophoresis using the Fragment Analyzer and the ‘Total RNA Standard Sensitivity Assay’ Results: The reads of all probes were adapter trimmed (Illumina TruSeq). Mapping was done against the Mus musculus (mm39; GRCm39) (June 24, 2020) genome sequence. After grouping of samples (four biological replicates each) according to their respective experimental condition, multi-group comparisons were made and statistically determined using DESeq2. The Resulting P values were corrected for multiple testing by FDR. A P value of <0.05 was considered significant. Conclusions: Our study represents the first detailed analysis of untreated and Beauvericin stimulated GM-CSF-cultured bone marrow derived cells transcriptomes.Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within GM-CSF-cultured bone marrow derived cells. We conclude that Beauvericin activates GM-CSF-cultured bone marrow derived cells inducing inflammatory cytokine,chemokine and Type I IFN production via a TLR4 dependent signaling pathway, but induces a gene expression profile different from LPS.
Project description:GM-CSF derived bone marrow cultures contain several subsets of CD11c+MHCII+ mononuclear phagocytes Using Affymetrix microarrays we compared gene expression of the different mononuclear phagocytes within the bone marrow culture. The different populations are left unstimulated or are stimulated with LPS The Affymetrix GeneChip Mouse Gene 1.0 arrays were used to define gene expression profiles in the different population.