(A) Graphed data of representative fluorescence-activated flow cytometry analysis of tnfa+ and tnfa− macrophages upon inflammatory stimulations. Tg(mpeg1:mCherryF/tnfa:eGFP-F) larvae were either kept intact (control), or amputated, or injected with PBS or with E. coli at 3 dpf, and cells were collected at 6 hr post-treatment. Red, green, and yellow gates represent mCherry+, eGFP+, and mCherry+eGFP+ populations, respectively. (B) Graph represents the kinetic of the frequency of mpeg1+tnfa+ macrophages in macrophage population (mpeg1+) in three independent experiments following stimulation: amputation and E. coli infection (E. coli) at indicated time points. *p < 0.05 vs 3 hpA, mean value of three experiments ±s.e.m. (C) Gating strategy to isolate control cells (mCherry− eGFP−, neg), tnfa− macrophages (mCherry+ eGFP−, mCh+), tnfa+ macrophages (mCherry+eGFP+, dbl+). (D–H) Relative expression of (D) mpeg1, (E) tnfa, (F) tnfb, il1b, (G) il6, (H) tgfb1, ccr2, and cxcr4b in cells neg, mCh+, and dbl+. Tg(mpeg1:mCherryF/tnfa:eGFP-F) were amputated at 3 dpf and cells were collected and separated at 6 hpA and 26 hpA. Real-time RT-PCR on separated cells using EF1a as a reference gene. Graph represents the mean value of five independent experiments ±s.e.m. Statistical significance between bars are indicated *p < 0.05, **p < 0.01.