After removing extra-intestinal fat tissue and blood vessels, colons were flushed of their luminal content with HBSS, opened longitudinally, and cut into 2 cm pieces and incubated with HBSS containing 0.015% DTT (15 min, 37°C in a shaking water bath). Epithelial cells and mucus were removed by extensive washes in HBSS, 5% FCS, 25 mM Hepes, before colons were cut into smaller 2mm2 pieces and digested in complete Iscoves media containing Liberase TL (167 µg/mL) and DNAse I (30 µg/mL) (Roche # 05401020001 and #10104150001) for 60 min at 37°C in a shaking water bath. The digested cell suspension was then passed through 100- and 40-µm cell strainers and resuspended in 1.077 g/cm3 iso-osmotic NycoPrep medium (Accurate Chemical & Scientific Corp.). After centrifugation at 1,600 g for 15 min, the low-density fraction was collected. Cells were then stained with Fc block [CD16/32 (2.4G2)] and antibodies to CD11c(HL-3), MHC-II (AF6-120.1), F4/80 mAb (BM8), CD11b (M1/70), CD103 (M290), as well as antibodies to B cell and T cell lineages (referred here after as “lin”): CD19 (RA3-6B2), CD3 (145-2C11), TCRβ (H57-597), TCRγδ (eBioGL3) all from eBioscience. After pre-gating on live, MHC-IIhigh lin- cells, the indicated dendritic cell and macrophage subsets were sorted using a BD Biosciences FACSVantage or FACSAria machine.
Growth protocol
Rag2–/– mice were injected i.p. with 1.5 x 105 CD4+ CD45RBhigh + 1.5 x 105 CD4+ CD45RBlow T cells (non-colitic CD45RBhigh+low mice), isolated from the spleens of wild type C57BL/6 mice.
Extracted molecule
total RNA
Extraction protocol
total RNA was isolated using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. RNA quantity /quality were assessed using the Agilent 2100 Bioanalyzer. RNA was reverse transcribed into cDNA (SuperScript III; Invitrogen)
Label
biotin
Label protocol
The cDNA was prepared and labeled according to standard manufacturer protocols (Affymetrix)
Hybridization protocol
Samples were hybridized onto Affymetrix GeneChip Arrays (Affymetrix) according to manufacturer's recommendations
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 7GPlus.
Description
Gene expression data from normal mouse macrophage sorted CD11cint F4/80hi
Data processing
The data were analyzed with GCOS 1.4 and EC 1.1 using Affymetrix default analysis and normalization method.
