Gene expression of resting memory, resting naive and in vitro activated memory CD8+CD127+ T cells from spleen and bone marrow (BM)

To understand differences between resting and activated memory CD8+ T cells, we compared the global gene expression of ex vivo isolated naive and spleen and BM memory cells to in vitro activated spleen and BM memory cells. Single cell suspension from the spleen and bones of aged C57BL/6 mice were prepared. Naive (CD44-CD127+) and memory (CD44+CD127+) CD8+CD3+ T cells were then cytometrically sorted. Sorted cells were either immediately processed for RNA preparation or were activated with anti-CD3 and anti-CD28 for 42-44 hours. Total RNA was extracted using the NucleoSpin RNA (Macherey-Nagel). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and 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_U430_2 GeneChips (Affymetrix) in triplicates. 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 All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.