Fig. 2.

Quantification of zebrafish neutrophils. All data presented here were collected from S. flexneri M90T-infected 2 dpf zebrafish larvae (2500 CFUs) at 2, 24 and 48 hpi, or from PBS-injected larvae. (A) Representative images of infected Tg(lyz::DsRed)^nz50 larvae. Assessment of neutrophil numbers in larvae at 48 hpi during emergency granulopoiesis is particularly challenging. Neutrophils are shown in white. Scale bar: 500 µm. (B) Comparison of leukocyte units/neutrophil numbers counted by hand (black), using the Ellett and Lieschke (E&L) method (green) or the LenCell macro (blue) in infected larvae at 2, 24 and 48 hpi. Leukocyte unit quantifications are provided. Data are presented without post-processing correction; n=44 per method. (C) Leukocyte unit quantification in infected larvae at 2, 24 and 48 hpi. Comparison between hand count of leukocyte units/neutrophils, and that using the Ellett and Lieschke method (green) or the LenCell macro (blue). Plotted values are the differences in percentage between the count using the macro and by hand for every sample (n=44 per method). **P<0.01; ****P<0.0001; ns, not significant. Two-way ANOVA with Sidak's multiple comparison test. Black bars: mean±s.e.m. (D) Correlation of leucocyte units between hand count (x-axis), and use of the Ellet and Lieschke method count (green) and the LenCell macro (blue) (both y-axis); n=132 per method. Black dotted line indicates the identity line. Green line indicates the linear regression on the Ellett and Lieschke method results (slope=1.561). Blue line indicates the linear regression of the LenCell macro results (slope=1.312).