title: colon_But_4
organism: Mus musculus
description: Protocols: Mice received an antibiotic cocktail containing ampicillin (1g/liter), metronidazole (1g/liter), neomycin (1g/liter), gentamicin (1g/liter), and vancomycin (0.5g/liter) in drinking water for 7 days, which was replaced once, after 3 to 4 days. Mice were administered acetate, propionate, or butyrate in their drinking water for 7 days at a concentration of 200 mM. SCFAs were added to LP cells in vitro at a concentration of 1 mM for 4 hours. These concentrations of SCFAs were based on published work (15, 17) and pilot experiments (no in vivo effects on macrophages <100 mM and decreased oral intake >300 mM; in vitro concentrations assessed for efficacy versus toxicity). The administered in vivo amount of 200 mM is higher than the physiological concentrations in the colon (20 to 50 mM) to take into account the absorption in the small intestine that will reduce the amount of SCFA reaching the colon. For in vitro experiments, 1 mM butyrate was used to take into account amounts of butyrate that might reach macrophages in the LP from the colonic lumen, where it is known that a substantial amount would be taken up by epithelial cells. Feces from control mice (housed separately from experimental mice) were administered to cages of both the experimental control group and the experimental antibiotic-treated group. Specifically, sawdust and feces from several cages of untreated (non-experimental) control mice were mixed together and distributed evenly between the experimental control group and the experimental antibiotic-treated group. Thus, mice were recolonized for variable time periods after 7-day antibiotic treatment. Cages and feces were replaced every 7 days. Intestinal macrophages were collected using an Aria Fusion FACS (fluorescence activated cell sorting) machine, from digested intestinal tissues. Macrophages were identified as CD45+ live CD11b+CD64+MHC Class II+ Ly6C- cells following neutrophil (Ly6G) and eosinophil (SiglecF) exclusion. RNA was extracted from samples using the Qiagen micro-Plus kit, following isolation of macrophages (via FACS) which were collected in RLT buffer. TruSeq® Stranded mRNA assay (Illumina, Inc.) according to the manufacturer's protocol
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-07-10T17:05:17Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-07-09T10:36:25Z
attributes.tag: External Id
attributes.value: SAMEA5763973
attributes.tag: INSDC center name
attributes.value: UNIVERSITY OF MANCHESTER
attributes.tag: INSDC first public
attributes.value: 2019-07-10T17:05:17Z
attributes.tag: INSDC last update
attributes.value: 2019-07-09T10:36:25Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7132:colon_But_4
attributes.tag: age
attributes.value: 10
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: macrophage
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: colon
attributes.tag: sample name
attributes.value: E-MTAB-7132:colon_But_4
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6