Gene expression profiling of murine plasmocytes isolated from the spleen of IL-10eGFP mice infected with Salmonella. Mus musculus

The goal of this study was to identify the molecular characteristics and putative markers distinguishing IL-10eGFP+CD138hi and IL-10eGFP-CD138hi plasmocytes. To this end, IL-10eGFP B-green mice were challenged intravenously with Salmonella typhimurium (strain SL7207, 10e7 CFU), and IL-10eGFP+CD138hi as well as IL-10eGFP-CD138hi plasmocytes were isolated from the spleen on the next day. For this, single cell suspensions were prepared, cells were treated with Fc block (10 µg/ml, anti-CD16/CD32, clone 2.4G2), and then stained with an antibody against CD138 conjugated to PE (1/400; from BD Pharmingen) followed by incubation with anti-PE microbeads (Miltenyi Biotech). CD138+ cells were then enriched on Automacs (Miltenyi Biotech) using the program possel_d2. Cells were then stained with anti-CD19-PerCP, anti-CD138-PE, and antibodies against CD11b, CD11c, and TCR conjugated to APC as a dump channel to exclude possible contaminants. DAPI was added to exclude dead cells. Live IL-10eGFP+CD138hi and IL-10eGFP-CD138hi cells were subsequently isolated on a cell sorter. The purity of the samples was always above 98%. This led to the identification of LAG-3 as a cell surface receptor specifically expressed on IL-10eGFP+CD138hi cells but not on IL-10eGFP-CD138hi cells. Overall design: Gene expression profiles of IL-10eGFP+CD138hi and IL-10eGFP-CD138hi cells. The experiment contains 2 groups. One group corresponds to IL-10eGFP+CD138hi cells, and the other group corresponds to IL-10eGFP-CD138hi cells. These cells were isolated from the spleen of IL-10eGFP mice (C57BL/6 genetic background) on day 1 after intravenous challenge with Salmonella typhimurium (strain SL7207, 10e7 CFU). The experiment was done in triplicates so there were three samples for each group. After total RNA extraction, reverse transcription, cDNA extraction, and in vitro transcription the biotinylated cRNA was fragmented, and hybridized on Affymetrix mouse genome 430 2.0 whole genome arrays. To obtain genes differentially expressed when comparing expression profiles of IL-10eGFP+CD138hi and IL-10eGFP-CD138hi cells group-wise comparisons were performed.