In vivo studies of the immune? activating functions of 5. (A) and (B) NF?B:eGFP induction by 5 in mpeg:mCherry positive macrophages in live zebrafish larvae. Zebrafish larvae (4?dpf) were stimulated with DMSO (control), R848 (positive control) or 5 for 6?h. A)?Representative confocal images of macrophages in live NF?B:eGFP/mpeg:mCherry transgenic larvae. Upper panel: DMSO control (arrow demarcates macrophage). Lower panel: 5 (B)?Flow cytometry analysis of NF??B induction in single?cell suspensions of zebrafish larvae. Cells were pre?gated on mpeg:mcherry macrophages and analysed for NF?B:eGFP expression. (C)?Quantitative real? time PCR for RNA expression of the inflammatory cytokines il1b, il6, tnfa in wildtype zebrafish larvae after 6?h activation with 5. DMSO was used as negative control. (n=3) *P<0.05; ** P<0.01 (D)?Il1b RNA expression in MyD88 mutant and wildtype zebrafish larvae after 6?h of stimulation with TLR ligands R848 and 5. DMSO was used as negative control. (n=4?5) Scale bars in A are 10??m.