Gene expression of Th cells, macrophages and monocytes derived from WT and Tbx21-/- Th cell-induced colitis

We compared gene expression profiles of Th cells, macrophages and monocytes isolated from the inflamed colon of colitis induced by the transfer of WT versus Tbx21-/- Th cells in Rag1-/- recipients. 4E05 naïve CD4+CD45RBhiCD25- Th cells from WT C57BL/6 or Tbx21-/- mice were transferred into congenic Rag1-/- recipients. When signs of colitis became apparent (day 15-17), a single cell suspension was prepared from the inflamed colonic lamina propria. Using a FACSAria sorter, live, single Th cells (CD3+CD4+), monocytes (CD64+Ly-6Chi) and macrophages (CD64+Ly-6Clow) were sorted. Th cells were stimulated for 3 hours in IMDM with 10% FCS containing PMA (10 ng/ml) and ionomycin (1 µg/ml). Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and amount was checked with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary RNA was synthesized from 1 µg total RNA using Message AmpII Biotin (Ambion, USA). Fifteen micrograms of fragmented cRNA of each sample were hybridized to MG 430_2 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.4, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.