Sample 4
Protocols: Mice were infected with 150 infective T. muris eggs in 200ul by oral gavage. Mice were culled at day 35 post infection via exposure to increasing carbon dioxide levels, an approved schedule 1 method as specified within the guidelines of the Animals (Scientific Procedures) Act. Cells were isolated as previously described with some modifications ( PubMed PMID: 17620362). In brief, after dissection of the caecum and colon, the colonic patch was removed from the tip of the caecum and all adherent adipose tissue removed from the tissue. The caecum and colon were cut longitudinally and washed thoroughly with pre-chilled PBS on ice. To remove intestinal epithelial cells and leukocytes, the tissue was incubated in prewarmed media (RPMI 1640) supplemented with 3% FCS (Sigma-Aldrich), 20 mM Hepes (Sigma-Aldrich), 5 mM EDTA (VWR, UK), and 1 mM freshly thawed dithiothreitol (Sigma-Aldrich) for 10 min at 37°C with agitation. After incubation, gut segments were shaken three times in fresh prewarmed RPMI 1640 (serum-free) with 2 mM EDTA and 20 mM Hepes. The remaining tissue was minced with fine scissors and digested at 37°C for 20 min with continuous stirring at 450 rpm in serum-free RPMI 1640 containing 20 mM Hepes, 0.15 mg/ml liberase TL (Sigma-Aldrich), and 0.5 mg/ml DNase I (Sigma-Aldrich). Digested tissue was passed sequentially through a 70-µm filter and 40-µm cell strainer, and after pelleting, it was resuspended in media supplemented with 10% FCS until staining. CD206+ and CD206- gut macrophages (gated as CD45+CD11b+CD11clow/intSiglecF-Ly6G-) were FACS-sorted on an Influx (BD biosciences) and isolated cells were suspended in PBS supplemented with 2% FCS (Sigma-Aldrich) and 2 mM EDTA (VWR). Sorted cells were collected in PBS with 10% FCS and stored on ice prior to washing and resuspending in Trizol (Thermo Fisher) before storage at −80°C for subsequent RNA extraction. Cells used for FACS-sorting were from a pool of two mice. Cell numbers after FACS sorting: Mouse 1+2 = 150,000 +ve, 150,000 -ve, Mouse 3+4 = 92,000 +ve, 116,000 -ve, Mouse 5+6 = 168,000 +ve, 132,000 -ve. All experimental procedures were approved by the University of Manchester Animal Welfare and Ethical Review Board and performed within the guidelines of the Animals (Scientific Procedures) Act, 1986. Male C57BL/6 mice were purchased from Envigo at age 6-8 weeks. Mice were maintained at a temperature of 20-22°C in a 12h light, 12h dark lighting schedule, in sterile, individually ventilated cages with food and water ad lib. RNA was extracted from sorted cells using an RNeasy mini kit (QIAGEN, UK) following the manufacturer's instructions. Truseq stranded mRNA Assay. Paired end.
ENA first public
2021-11-15
ENA last update
2021-11-15
External Id
SAMEA9067783
INSDC center alias
UNIVERSITY OF MANCHESTER
INSDC center name
UNIVERSITY OF MANCHESTER
INSDC first public
2021-11-15T12:29:33Z
INSDC last update
2021-11-15T12:29:33Z
INSDC status
public
Submitter Id
E-MTAB-10501:Sample 4
age
11 to 13
broker name
ArrayExpress
cell type
gastrointestinal tract (lamina propria) macrophage
common name
house mouse
developmental stage
adult
genotype
wild type genotype
individual
1 and 2
infect
Trichuris muris
organism part
mixed caecum and colon
phenotype
CD206 positive
sample name
E-MTAB-10501:Sample 4
sex
male
strain
C57BL/6