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Fig. 3
Neutrophils are increased in AB but not in TU after TNBS exposure. AB and TU were crossed with LysC;Dsred + zebrafish the Dsred + progeny was selected (LysC:AB and LysC:AB) and exposed to TNBS (T70). At 6 dpf analysis of intestinal immune cell infiltrate was analyzed by flow cytometry (A and B) or microscopy (C and D). For FACs analysis intestines of LysC:AB and LysC:AB were dissected and dissociated; (A) a gate on granulocytes was performed based on size and granularity and using cells isolated from the hematopoietic organ (kidney) of an adult zebrafish as reference and no significant differences between LysC:AB and LysC:TU was found; (B) the frequency of neutrophils (DsRed + cells) was unaltered in LysC:TU after TNBS exposure compared to control and in LysC:AB mean value increased compared to control but was not statistically significant. Each sample is a pool of 100–200 larval intestines, data is representative of three independent experiments. On another set of experiments, microscopic quantification of neutrophils in the gut of control or TNBS-exposed (T70) larvae was performed; (C) representative images of larvae showing delimitation of gut area (inside dotted lines) can be seen; (D) our data shows that in physiological conditions there are no differences between LysC:AB and LysC:TU and that TNBS exposure significantly increases the number of neutrophils in gut LysC:AB compared to control &&P ≤ 0.01 and vs. T70 LysC:TU, **P ≤ 0.01. In LysC:TU, TNBS caused a small but significant decrease in neutrophils compared to control #P ≤ 0.05. Each point represents 1 animal, for each group 9–13 animals were evaluated, data is representative of two independent experiments. Data are presented as mean and standard error of mean and differences were calculated by unpaired t-student test.