Briefly, total RNA was isolated from the cells with an ArrayGrade total RNA isolation system, then purified using a spin column (SA Biosciences). The purity and quantity of the extracted RNA were checked with Nanodrop. A total of 1·5 μg RNA was reverse transcribed to cDNA, followed by real-time PCR (One step; Applied Bioscience, Foster City, CA) and data analyses was performed using the SA Bioscience Array expression analysis suite. Terminal ileums were excised from SAMP1/Yit and AKR/J
mice of various ages, then immersion-fixed in 10% formaldehyde for 48 hr. Idasanutlin order Next, the tissues were embedded in paraffin and cut into 6-μm sections, and stained with haematoxylin & eosin to visualize the general morphology under a phase contrast light microscope. To verify the role of MLN B cells in IL-1β production by TLR-mediated macrophages, we conducted an in vitro experiment. LDK378 order Peritoneal macrophages (1 × 106 cells/well) isolated from AKR/J and SAMP1/Yit mice were co-cultured with purified MLN B cells
(1 × 106 cells/well) from SAMP1/Yit or AKR/J mice in 24-well plates, then stimulated with LPS (100 ng/ml) or CpG-DNA (100 nm/ml) for 72 hr. IL-1β contents in the culture supernatants were examined by EIA. To understand the role of MLN B cells in IFN-γ production by TLR-mediated intestinal T cells, we conducted an in vitro experiment. MLN T cells (1 × 106 cells/well) check details isolated from AKR/J and SAMP1/Yit mice by using the pan-T-cell-specific marker CD90.1 microbeads were co-cultured with purified MLN B cells (1 × 106 cells/well) from
both mice in 24-well plates, then stimulated with LPS (100 ng/ml) or CpG-DNA (100 nm/ml) for 72 hr. The IFN-γ content in the culture supernatants was examined by EIA. All data are expressed as the mean ± standard error of the mean (SEM). Values were analysed using Student’s t-test and Spearman’s rank correlation with Stat-View 4.0 software (Abacus Concepts, Inc., Berkeley, CA). For comparisons of multiple values, analysis of variance was used. P values < 0·05 were considered significant. Initially, we used BALB/c mice and examined cell surface markers of B cells isolated from several parts of the mice using flow cytometry, with representative results shown in Fig. 1. In the B cells isolated from the MLNs, PPs, colon lamina propria, and spleens, similar expression patterns of CD1dhigh, CD5low, CD11b−, TLR4/MD-2low and TLR9low were observed. In contrast, high expression levels of CD5, CD11b and IgM were found in B cells isolated from PerC. We also noticed a significant expression of RP105 in B cells isolated from various organs. RP105, which is associated with MD-1 protein, was the first leucine-rich repeat (LRR) protein found on the surface of B cells.