ABSTRACT: Macrophages and dendritic cells (DC) are key components of cellular immunity and, as per the current model, originate from hemopoietic stem cells (HSCs). However, microglia were recently shown to originate from precursors that precede the appearance of HSCs. We thus re-investigated macrophage development and their relationship to HSCs and yolk sac (YS) precursors.
Project description:Six-weeks old (C57Bl6, Cx3cr1gfp/+) mice were intraperitonealy infected with a low number (1.104) of L. monocytogenes (EGDe strain) in exponential growth phase (bacteria were grown in BHI at 108/ml, and diluted 10.000x in PBS immediately before injection). Group of three mice were euthanized, before infection. Peritoneal cells were recovered by peritoneal lavage. Cells from individual mice were stained with antibodies to CD11b (PECy7), Gr1 (APC), NK1.1, B220 and CD3 (PE), and F4/80 (biotin-conjugated followed by streptavidinpacific blue) for sorting. Gr1- monocytes were purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1-, gfphigh; Gr1+ monocytes were purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1+, gfpint; and polymorphonuclear cells were purified as NK1.1- CD3- B220- CD11b+ F4/80- Gr1high, gfp-. 1.103 cells from each mice, time point, and phenotype were purified by facs sorting according to their phenotype. Samples were kept at 4°C before and during the sort. Cells were directly sorted in the SuperAmp Lysis Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) using a FACS Aria cell-sorter (BD biosciences).
Project description:Comparison of gene expression profile of different types of cells: hESCs (H1), human adult dermal fibroblasts, cells from adult human ovarian cell culture, FACS sorted SSEA-4 positive cells from adult human ovarian culture and cells from human ovarian cancer cell culture.
Project description:In vivo studies in mice have identified subsets of monocytes involved in the inflammatory response to pathogens, but their respective functions remains poorly understood. We report that human CD14+ monocytes represent functional ortholog to mouse Ly6C+ monocytes and are responsible for phagocytosis, Reactive Oxygen Species (ROS) and cytokine production in response to a broad range of pathogen associated molecular patterns (PAMP). In contrast a discrete population of CD14dim cells are ortholog to the mouse ly6C- monocytes and share their ability to patrol the endothelium of blood vessels, but do not produce ROS, are weak phagocytes, and poorly respond to bacterial PAMPs. Instead, CD14dim monocytes are critical for production of pro-apoptotic and pro-inflammatory cytokines in response to viruses and nucleic acids via a unique TLR7-8/MyD88/MEK pathway, while they may also contribute to tissue damage in autoimmune diseases such as Lupus.
Project description:Human monocytes are phagocytic leucocytes which circulate in the peripheral blood and play important roles in immunity and inflammation. They also represent circulating M-^QmetabolicM-^R sentinels in plasma, uptake lipids following food intake, and contribute to atherosclerotic plaque formation. We therefore investigated their homeostatic responses to food intake in vivo and to determine the response of human monocytes to food intake. We isolated monocyte subsets from the blood of healthy volunteers one hour before and four hours after a western type lunch.
Project description:The aim of this study was to analyze the global transcriptional profiles of small intestine (SI) Innate Lymphoid Cells (ILCs) expressing the NK cell marker NKp46. Based on differential expression of the RORgt transcription factor SI NKp46+ ILCs can be divided in NKp46+RORgt- and NKp46+RORgt+ cells. While NKp46+RORgt- cells produce IFN-g, like conventional Natural Killer (NK) cells, NKp46+RORgt+ cells secrete IL-22, like Lymphoid Tissue inducer (LTi) cells. We compared the global transcriptional profiles of both NKp46+RORgt- and NKp46+RORgt+ cells to conventional splenic NK cells and to SI NKp46-RORgt+ cells, which contain adult LTi cells. By following this approach, we showed that SI NKp46+RORγt- ILCs correspond to SI NK cells. We also identified a transcriptional program conserved in adult SI NKp46+RORγt+, NKp46-RORγt+ ILCs and fetal LTi. The various ILC cell populations analyzed in this study were isolated from C57BL/6 RORc(gt)+/GFP reporter mice. SI NKp46+RORγt- (NKp46+GFP-) cells, SI NKp46+RORγt+ cells (NKp46+GFPlow and NKp46+GFPhigh cells) and NKp46-RORγt+ ILCs, including adult LTi cells , were sorted by flow cytometry from CD3- lamina propria cells of small intestine (SI) of RORc(γt)+/GFP reporter mice . Splenic NKp46+RORγt- (NKp46+GFP-) cells were also sorted as the reference for conventional NK cells. Two replicates of each populations were produced and analyzed.
Project description:CD56brightCD16-, CD56dimNKG2A+ and CD56dimNKG2A- NK cells subsets were FACS sorted and their transcriptional profile were compared after whole genome microarray analysis.
Project description:To evaluate geneexpression profile in developing joints vs adjacent growth plate in control and TGF-beta type II receptor conditional knock-out in limb mesenchyjme We obtained E14.5 autopods from two normal controls and two Tgfbr2Prx1KO littermate embryos. Samples were frozen in OTC and cryosectioned and subjected to laser-capture microdissection for RNA sampling for interzone, growth plate chondrocytes and interdigital tissue. RNas were subjected to microarray analysis
Project description:Transcriptional profiling of Langerhans cells isolated from CD11c-Cre x Dicer wt/wt (Dicer +/+ ) and CD11c-Cre x Dicer fl/fl (Dicer-/-) mice. Langerhans cells are isolated via enzymatic digestion of skin followed by antibody staining and FACS sorting
Project description:Analysis of Hoechst dye 33342-effluxing side population (SP) cells from B-CLL peripheral blood mononuclear cells. 9 biological replicates from B-CLL patients sorted into CD5+CD19+ SP and non-SP subsets. Two color comparative gene expression using Agilent microarrays.