Gene expression of murine IgG2-memory B cells from spleen and bone marrow

We compared gene expression profiles of murine IgG2b+ memory B cells isolated from spleen and bone marrow of C57BL/6J mice and mice obtained at a pet shop. 6x10E04-1.5xE05 IgG2b+ memory B cells from spleens and bone marrow of WT C57BL/6 J Crl mice or mice obtained at a pet shop in Berlin, Germany were isolated using a FACSAria II sorter following magnetic enrichment of CD19+ cells. A single cell suspension of spleen or bone marrow cells was prepared, CD19+ cells isolated by MACS technology (Miltenyi Biotec) and then FACSorted for IgG2b+IgM-IgD-CD19+CD38+CD138-CD11c-GL7-PI- small lymphocytes. Total RNA was extracted using the RNA MiniPrep kit (Zymo Research). 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 All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.