The Nod2 Agonist Muramyl Dipeptide Cooperates with the TLR4 Agonist Lipopolysaccharide to Enhance IgG2b Production in Mouse B Cells

Figure 5

Effects of LPS and MDP on cell viability, proliferation, and Ab production and germline transcripts expression in TLR4- and Nod2-deficient B cells. Resting B cells were purified from wild-type (WT), TLR4-deficient (Tlr4^-/-), and Nod2-deficient (Nod2^-/-) B cells and stimulated with MDP (10 μg/mL) and iE-DAP (10 μg/mL) in the presence or absence of LPS (1 μg/mL). (a) After 2 and 3 days of culture, cell viability (OD) was measured by EZ-Cytox assay kit, and cell proliferation was measured by CFSE assay. Low CFSE intensity cell (%) means the proportion of proliferating cells. (b) After 7 days of culture, supernatants were harvested, and the levels of Ab production were measured by isotype-specific ELISA. Data shown are averages of triplicate cultures with SEM error bars. SEM: standard error of the mean. . (c) After 2.5 days of culture, RNAs were isolated and the levels of germline transcripts and AID mRNA were measured by RT-PCR. The levels of germline transcripts and AID mRNA were measured by semiquantitative RT-PCR with 1/5 and 1/25 diluted cDNA (c, lower panel). The graphs show relative GLTγ2b level normalized to β-actin cDNA expression using ImageJ, and data are averages of two independent experiments with ranges (bars).

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