Colon IEL gene expression

Comparison of gene expression by colon IELs versus small intestine IELs and colon lamina propria T cells T cells from the colon epithelium, lamina propria, and small intestine epithelium were magenetically enriched by negative selection from C57Bl/6 mice ages 8-10 weeks. 10 total mice were pooled (5 male and 5 female). One group of mice were treated with broad-spectrum antibiotics (vancomycin, ampicillin, neomycin, and metronidazole) for one week then recolonized by co-housing with unmanipulated littermates for one week prior to harvesting the colon IELs. Four samples were analyzed for gene expression by microarray: Colon lamina propria T cells (LP), colon intraepithelial lymphyocytes from unmanipulated mice (WT_IEL), colon IELs from mice treated with antibiotics followed by bacterial recolonization (Recolonized_IEL), and IELs from the small intestine of unmanipuatled mice (SI_IEL). Methods for the acquisition of these cell types are as follows: A total of 10 C57Bl/6 mice (5 males and 5 females) at 8-10 weeks of age were pooled for each sample. Mice were treated with 1% grape flavored Koolaid (Kraft), 0.01% ampicillin, 0.01% neomycin, 0.05% metronidazole (Research products International), and 0.05% vancomycin (Alfa Aesar) delivered ad libitum in the drinking water for 7 days. Re-colonization of antibiotic-treated SPF housed animals was done using mixed dirty bedding from untreated littermates for 7 days. Mice were then euthanized and colon and small intestine (SI) harvested. Tissue was minced and shaken at maximum speed on a benchtop vortex for ten minutes in 10ml PBS (Gibco) supplemented with 1 mM EDTA and 7.5 mM HEPES to isolate the epithelial fraction. Cells were passed through a 70um cell strainer to collect the epithelial fraction containing intraepithelial lymphocytes (IELs). Lamina propria cells were isolated after epithelial-isolation. Tissue was finely minced and incubated for 30 minutes at 37C in 0.5 mg/ml type IV collagenase (Worthington) in RPMI 1640 (Gibco) supplemented with 10% FBS (Thermo), 1mM EDTA, and 7.5mM HEPES. Digested tissue was passed through a 70um cell strainer (Corning). T cells from the SI epithelial fraction, colon epithelial fraction, and colon lamina propria fraction through magnetic enrichment using the EasySep Negative T cell isolation kit (Stemcell) following the manufacturer s recommended protocol with the addition of 0.5 ng/mL biotin-labeled anti-mouse EpCAM (clone G8.8, Biolegend).