RNA-seq of sorted mouse gdT cell populations based on TCR Vg chain and Ly6C status
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ABSTRACT: Gamma-delta (gd) T cells from pooled mouse lymph nodes and spleens were isolated and FACS-sorted for Vg1+Ly6C-, Vg1+Ly6C+, Vg4+Ly6C- and Vg4+Ly6C+ subsets of bulk CD27+ gdT cells.
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:Macrophages are a heterogeneous cell type implicated in injury, repair, and fibrosis after AKI, but the macrophage population associated with each phase is unclear.results of this study in a renal ischemia-reperfusion injury model allow phenotype and function to be assigned to CD11b+/Ly6C+ monocyte/macrophage populations in the pathophysiology of disease after AKI. we used a renal bilateral ischemia-reperfusion injury mouse model to identify unique monocyte/macrophage populations by differential expression of Ly6C in CD11b+Â cells and to define the function of these cells in the pathophysiology of disease on the basis of microarray gene signatures and reduction strategies Macrophage populations were sorted by Flow Cytometry into low and intermediate populations by Itgam(Cd11b) and Ly6c markers. The cells obtained in 5 weeks sham, 5 weeks IR, 9 day sham, and 9 day IR with 6 samples per group (3 int and 3 low). Cells were sorted in 350ul of RLT lysing buffer and kept at -80c until RNA extraction.Sample amplification, fragmentation, hybridization,washing and scanning were performed according to validated Affymetrix protocol in a CLIA certified lab.
Project description:In this study we investigated the mechanisms involved in memory T-cell mediated protection using mice vaccinated with the intracellular bacterium Listeria monocytogenes. Our working hypothesis was that rapid activation of cells of the innate immune system, in particular inflammatory Ly6C+ monocytes, were essential in effective protection, in a memory T cell-dependent manner. Thus we generated a comprehensive comparison of the genetic program of activated Ly6C+ monocytes during a primary or a secondary infection with Listeria monocytogenes, at 8 hours post challenge infection. Abstract of corresponding publication: Cells of the innate immune system are essential for host defenses against primary microbial pathogen infections, yet their involvement in effective memory responses of vaccinated individuals has been poorly investigated. Here we show that memory T cells instruct innate cells to become potent effector cells in a systemic and a mucosal model of infection. Memory T cells controlled phagocyte, dendritic cell and NK or NK T cell mobilization and induction of a strong program of differentiation, which included their expression of effector cytokines and microbicidal pathways, all of which were delayed in non-vaccinated hosts. Disruption of IFN-gamma-signaling in Ly6C+ monocytes, dendritic cells and macrophages impaired these processes and the control of pathogen growth. These results reveal how memory T cells, through rapid secretion of IFN-gamma, orchestrate extensive modifications of host innate immune responses that are essential for effective protection of vaccinated hosts. Overall design: Inflammatory monocytes were purified (see below for isolation method) from 4 groups of 3 individual mice each (triplicate): (i) uninfected mice, (ii) primary infected, (iii) secondary infected, (iv) secondary infected and T-cell depleted 1 day before. Isolation of cells was done on 3 different days for true biological replicates.
Project description:Muscle injury was elicited by cardiotoxin injection into the tibialis anterior muscle. Macrophages were isolated 2 days post-injury from the regenerating muscle. We used microarray to obtain global gene expression data of muscle-derived tissue macrophage subsets. Tissue macrophages were collected from regenerating muscle samples of three animals, Ly6C+ F4/80low and Ly6C- F4/80high macrophage subsets were sorted. The global gene expression patterns of distinct macrophage subsets were analyzed on Affymetrix microarrays.
Project description:Muscle injury was elicited by cardiotoxin injection into the tibialis anterior muscle. Macrophages were isolated 2 days post-injury from the regenerating muscle. We used microarray to obtain global gene expression data of muscle-derived tissue macrophage subsets. Tissue macrophages were collected from regenerating muscle samples of three animals, Ly6C+ F4/80low and Ly6C- F4/80high macrophage subsets were sorted. The global gene expression patterns of distinct macrophage subsets were analyzed on Affymetrix microarrays.
Project description:Neutrophils (single cells - Ly6G+ cells) from 4T1 tumor bearing mice were sorted based on Ly6C high or low expression and sequenced.
Project description:Purpose: To compare the transcriptomes of activated CD4 T effector cell populations in the presence and absence of STAT3 at 8 days post-infection using high-throughput RNA sequencing analysis. Methods: Cell sorting of the populations was done using the markers Ly6c and PSGL-1 CD4 T cell Ly6c and PSGL-1 population mRNA profiles 8 days post-LCMV infection of wild type (WT) and STAT3fl/fl Cd4cre mice were generated by mRNA sequencing using Illumina HiSeq 2000.