Figure 6.

B cell-activating factor (BAFF) increases the number of IgM-secreting cells. (A) Splenocyte cultures were treated with BAFF (3 μg/ml), TNP-LPS (5 μg/ml), a combination of both, or left unstimulated (control) for 48 h and then plated in ELISPOT plates previously coated with anti-trout IgM mAb, for a further 24 h. After incubation, cells were washed away and a biotinylated anti-trout IgM mAb was used to detect numbers of spot forming cells. Quantification of spot forming cells is shown as mean + SD (n = 12, from four independent experiments containing three animals each). (B) The number of IgM-secreting cells was also plotted for each individual fish under control or BAFF stimulation conditions. (C) Splenocyte cultures were treated with BAFF (3 μg/ml) or left unstimulated for 24 h and, then, RNA from IgM⁺ FACS-isolated B cells was extracted as described in Section “Materials and Methods.” The transcription of Blimp-1 relative to the endogenous control EF-1α was calculated for each sample and shown as mean + SD (n = 12, from four independent experiments containing three animals each). Statistical differences were evaluated by a one-way ANOVA followed by a multiple comparison Tukey’s test, where **p ≤ 0.01.