Immunoglobulin class-switch recombination downregulates Blimp1-driven IL-10 production in IgM+CD138hi cells

To determine characterize IL-10 producing CD138hi cells, we compared the global gene expression of primary B220+ B cells in spleen, freshly isolated Venus+ CD138hi cells in bone marrow and spleen of IL-10 Venus reporter mice. B220+ and Venus+ CD138hi cells from spleen and bone marrow of IL-10 Venus mice were sorted by flow cytometric cell sorting on a BD Aria III sorter (Becton Dickinson). Total RNA was purified using RNeasy Plus Mini Kit (QIAGEN). Total RNA was labeled with Cy5 using the Amino Allyl Message AMP II aRNA Amplification Kit (Applied Biosystems). The Cy5-labeled aRNA was fragmented and then hybridized to 3D-Gene Mouse Oligo chip 24k (Toray Industries Inc.). Hybridization signals were scanned with a 3D-Gene Scanner and processed by 3D-Gene Extraction software (Toray Industries). The raw data of each spot were normalized by subtraction with a mean intensity of the background signal determined by all blank spots' signal intensities of 95% confidence intervals. Detected signals for each gene were normalized by a global normalization method (the median of the detected signal intensity was adjusted to 25)