Figure 2.

Short, but not prolonged treatment of salmon MPs with CpGs upregulates their allostimulatory activity. (A), adherent HK MPs were stained with CFSE upon 24 h of stimulation with 2 μM CpGs. Splenocytes stained with CellTrace Violet (CTV) were used as responders. The dot plots show that a significant number of stimulators take up CTV which necessitates their staining with CFSE in order to distinguish them from proliferating responders with reduced CTV staining. The CFSE-negative events were gated and displayed in histograms to quantify the percentage of proliferating splenocytes with reduced CTV staining. In the representative histogram (bottom right), responders were cultured with non-stimulated (NS) MPs from another individual (filled gray area), and stimulators pretreated with CpGs for 24 h (black contour). (B), Stimulators pretreated with CpGs for 24 h but not 7 days show increased allostimulatory capacity (n = 5). Autologous stimulators did not significantly activate splenocyte proliferation even after pretreatment with CpGs for 24 h (*p < 0.05). (C), Direct CpG stimulation of responders was included as a positive control for cell proliferation (n = 4). In all of the experiments, the responders were cultured in vitro for 15 days.