Project description:To probe potential differences between awake and anesthesia microglia, we performed the single-cell RNA sequencing (scRNA-seq) . Microglia were isolated by FACS as CD11b high CD45 low cells from awake and anesthesia mouse brains.
Project description:To probe potential molecular mechanisms underlying the differences between wild-type and Hk2-cKO microglia during repopulation, we performed the single-cell RNA sequencing (scRNA-seq) of repopulated microglia at Day 3. Microglia were isolated by FACS as CD11b high CD45 low cells from normal brains and Hk2-cKO brains.
Project description:We used single-cell RNA sequencing (scRNA-seq) to analyze the diversity of HSPC-derived CD45+CD11b+ microglia-like cells (MGLCs) engrafted in the brain of recipient mice that were conditioned using Busulfan and PLX3397 and transplanted intravenous with Lineage negative KIT+ SCA1+ mouse HSPCs. The HSPCs were isolated from adult C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ homozygous mice. We compared the gene expression of MGLCs to that of developmentally-derived CD45+ CD11b+ microglia/myeloid cells isolated from the brain of recipient mice (host microglia) and untreated mice (naive microglia). We also compared the gene expression of MGLCs to that of transplant-derived CD45+ CD11b+ cells engrafted in the bone marrow (abbreviated as BM-CD11b+)
Project description:We used single-cell RNA sequencing (scRNA-seq) to analyze the diversity of bone marrow-derived CD45+CD11b+ microglia-like cells (MGLCs) engrafted in the brain of recipient mice that were conditioned using Busulfan and PLX3397 and transplanted with total bone marrow. We compared the gene expression of MGLCs to that of developmentally-derived CD45+ CD11b+ microglia/myeloid cells isolated from the brain of recipient mice (host microglia) and untreated mice (naive microglia). We also compared the gene expression of MGLCs to that of transplant-derived CD45+ CD11b+ cells engrafted in the bone marrow (abbreviated as BM-CD11b+)
Project description:Gene profiling of CNS-derived microglia vs splenic CD11b+Ly6C+ monocyte subsets deom adult mice Gene array identified 1572 genes that were enriched in microglia vs. 611 monocyte enriched genes with a greater than 5-fold difference (P<0.001). Gene profiling of CD11b+CD45Low microglia isolated from the CNS and CD11b+Ly6C+ monocyte subsets isolated from the spleen of naM-CM-/ve adult mice.
Project description:To capture the global gene expression patterns in the CECs and microglia following acute systemic inflammation, we injected the mice with LPS and sacrificed them after 30 min, 1 hr and 2 hrs, immediately followed by the isolation of the brains and dissociation to yield single cell suspension, immunolabelling and finally flow assisted cell sorting of CECs (CD45- CD13- CD31+) and microglia (CD45 low-mid CD11b+). RNA was then isolated from the cells followed by RNA-seq.
Project description:Microglia isolation from adult mouse brains remains difficult in comparison to microglia isolation from brains from newborn mouse brains as connective tissue, extracellular matrix materials and thicker myelin sheaths substantially influence traditional isolation protocols used with newborn mouse brains. Since the introduction of magnetic activated cell sorting (MACS), it has been reported that it is possible to also obtain microglia cells from adult mouse brains with a very good purity. However, most studies only used flow cytometry and/or qPCR to determine microglia enrichment and enrichment of traditional microglia marker proteins. Therefore we wanted to characterize in an unbiased way the proteomes of with MACS isolated microglia cells (CD11b positive cells) in comparison to all non-CD11b positive cells (termed non-target cell fraction (NTCF)), which consist of primarily astrocytes, neurons and oligodendrocytes. Therefore, we isolated from three adult mouse brains via MACS CD11b positive cells as well as the remaining CD11b negative cells (NTCF) to characterize the proteome of both cell fractions. The obtained proteomes were compared to a dataset of proteomic signatures for all CNS cell types determined by Sharma et al., (2015, doi: 10.1038/nn.4160) to determine the amount of enrichment of microglia-specific proteins.
Project description:Mice were generated that had a tamoxifen-inducible Cre recombinase transgene under the control of the CX3CR1 promoter. These mice were crossed with mice that had a floxed PPAR-delta allele. EAE was induced in these mice and controls that just had the floxed allele at 30 days after tamoxifen treatment. Mice were euthanized (N=9/genotype) for isolation of microglia at 2-3 days after the onset of EAE, a time when clinical scores were equivalent between the groups. Spinal cords were pooled (N=3/sample) for microglia isolation and sorting. Microglia were isolated from spinal cords by collagenase digestion, percoll gradient and FACS sorting using CD45 and CD11b antibodies resulting in N=3 spinal cords resulting in N=3 pooled samples/group. RNA was isolated from sorted microglia using the PicoPure™ RNA Isolation Kit (Thermo Fisher Scientific). RNA quality was evaluated using the 2100 Bioanalyzer (Agilent) and N=2 samples per group with RIN numbers > 7 were submitted for sequencing and analysis at the UHN Bioinformatics and HPC Core, Princess Margaret Cancer Centre.
