Gene expression of murine long-lived bone marrow plasma cells ex vivo and plasma cells cultured for 3 days with ST2 stromal cells and the cytokine APRIL under physiological oxygen conditions.

We compared gene expression profiles of murine longlived bone marrow plasma cells isolated from immunized C57BL/6J in the memory phase with plasma cells co-cultured for 3 days with murine ST2 stromal cells in the presence of the cytokine APRIL under physiological oxygen conditions. C57BL/6J mice were primed with NP-CCG in incomplete Freud`s Adjuvants intraperitoneally. Mice were challenged twice after the prime with the same injection in cycles of 21 days. 30 days after the last boost, longlived plasma cells were magnetically isolated (Miltenyi Biotec, Germany) with a 2 step-protocol including depletion of B220 and CD49b expressing cells and subsequent positive enrichment of CD138high plasma cells. Plasma cells were directly processed for RNA preparation or cultured for 3 days in co-culture with murine ST2 stromal cells in the presence of the cytokine APRIL under physiological oxygen conditions. After culture, plasma cells were FACSorted using FACSAria II. Total RNA was extracted using the RNeasy Micro KIT (Quiagen) and hybridized to MG 430 2 GeneChips according to the whole-transcriptome pico KIT. 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). 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.