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Fig. 4 Quantitative analysis of PLA-NP internalization by macrophage from 10 min to 24 h post-injection. (A) Automated analysis of the relative amount of internalized fluorescent PLA-NP by macrophage 10 min and 24 h following intravenous injection of 3 dpf mpeg1:mCherry zebrafish. Stereo-microscope acquisitions of individual zebrafish were put through an ImageJ macro, from which an internalization score was calculated, based on the amount of red PLA-NP fluorescence associating with the green macrophage fluorescence signal, relative to the total amount of red PLA-NP fluorescence detected from the whole fish. Histograms represent the mean internalization score and each dot represents the score of an individual zebrafish. From 30 min to 24 h post-injection, more NP were internalized by macrophages (+56%). (B) Flow cytometry analysis of the fraction of macrophages that had internalized fluorescent PLA-NP relative to the whole population of macrophages, 10 min to 24 h after intravenous injection into 3 dpf zebrafish. Histograms represent the mean percentage of PLA-NP positive macrophages at different time-points following administration, with each dot representing the data acquired from 10 pooled larvae. Almost 8% of macrophages were labelled positive for PLA-NP 10 min after the injection. After this time, the fraction of macrophage internalizing PLA-NP steadily increased over time until it reached a plateau around 17% of PLA-NP-positive macrophages at 4 h and 24 h post-injection. All the flow-cytometry acquisitions are illustrated in (B?), they only display the population of macrophage which was gated based on the mCherry fluorescence intensity. The x axis displays the ?size? of the detected events and the y axis the fluorescence intensity corresponding to PLA-NP. Error bars: SEM. Significance level is indicated as: **p < .01. Number of fish per condition: (A) 30 min (36) and 24 h (40); (B) 10 larvae per replicate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)