Fig 3

(A) qRT-PCR of pro-inflammatory mRNAs from sorted neutrophils from 6 dpf WT and saa mutant zebrafish larvae (5,000–12,000 lyz:EGFP⁺ cells / replicate, 3–6 replicates / genotype / experiment, 60–90 larvae / replicate). (B) Microscopic analysis revealed neutrophils isolated from adult zebrafish kidneys extend protrusions in response to bacterial signals ex vivo (white arrows; scale bar = 20 μm). (C)il1b expression in un-stimulated and E. coli exposed lyz:EGFP⁺ neutrophils from WT and saa mutant zebrafish following 4 hours ex vivo culture (3–5 replicates / genotype / condition). (D) CFU quantification of bacterial concentration following 4 hour co-culture of isolated lyz:EGFP⁺ neutrophils from WT and saa mutant zebrafish with E. coli (MOI 2). (E) Quantification of intracellular ROS levels by CellROX fluorescence from neutrophils cultured ex vivo with and without E. coli (lyz:EGFP⁺ cells isolated from 6 zebrafish adult kidneys / genotype, quantification of ≥ 177 cells / condition). In panels C and E, a one-way ANOVA with Tukey’s multiple comparisons test was used. Data in panels A and D were analyzed with a t-test. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.