Figure 3. Quantification of platelet recruitment to livers of mice treated with intravenous LPS using CD41-YFPki/+ mice. Representative fields of view of livers from untreated (Ai, Aii) and LPS treated (Bi, Bii) CD41-YFPki/+ mice. Neutrophils are labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue); YFP+ platelets and liver autofluorescence (green). All scale bars, 20 mm. (C) Quantification of the number of adherent (stationary for $30 s) and interacting (present in the field of view for but not stationary for $30 s) YFP+ platelets per field of view of liver in untreated and LPS treated CD41-YFPki/+ mice. (D) Average number of YFP+ platelets adherent to each neutrophil within the livers of untreated and LPS treated CD41-YFPki/+ mice. (E) Percentage of neutrophils with zero or with three or more adherent platelets within the livers of untreated and LPS treated CD41-YFPki/+ mice. *** p,0.001. doi:10.1371/journal.pone.0025109.g003 numbers of labelled platelets. Mice used in this study had an average of 21.6%65.9% YFP positive platelets. Intravital observation of YFP+ platelets in the liver sinusoids of untreated mice reveals little platelet adhesion or aggregation (Fig. 3Ai, 3Aii), an observation similar to what was seen when anti-CD49b was used to label the platelets. Some YFP+ platelets appear to interact with the sinusoidal walls or adherent neutrophils, touching down briefly, but then quickly releasing Intravital Visualization of Platelets and re-entering the blood flow (Movie S9), a behaviour that is again consistent with that observed when platelets were labelled with anti-CD49b. In contrast, livers of mice treated with LPS demonstrate pronounced platelet recruitment (Fig. 3Bi, 3Bii), where YFP+ platelets are seen to be associated with the sinusoid walls and even more so with the numerous recruited neutrophils (Movie S10). It is important to note that only approximately 20% of platelets are visible in these mice and, as such, visualization of platelet-platelet interactions and platelet aggregation is limited. Furthermore, because not all platelets are labelled, it is impossible to measure the total size of platelet aggregates in this model. One advantage of these mice, however, is individual platelets can be visualized more easily, thus making it easier to study platelet behaviour on the level of the single platelet. Quantification of the number of adherent (stationary for $30 s) and the number of interacting platelets (present in a field of view for $30 s but not stationary) demonstrates significant increases in LPS-treated mice when compared to untreated mice (Fig. 3C). Furthermore, we can characterize the interactions of individual platelets with adherent neutrophils in the liver. Following LPS treatment, the average number of YFP+ platelets bound by each neutrophil (platelets that remain adherent for $30 s) increased significantly as compared to untreated mice (Fig. 3D). Moreover, the percentage of neutrophils that were not associated with YFP+ platelets was significantly reduced and the percentage of neutrophils binding three or more YFP+ platelets increased significantly following treatment with LPS (Fig. 3E). These CD41-YFPki/+ data support the observations made using anti- CD49b where a 3-4 fold increase in the total number of platelet aggregates ($10 mm2, Fig. 2Di), many of which are adherent to neutrophils, is seen in the livers of mice treated with LPS. A more detailed understanding of platelet recruitment to the liver following LPS exposure can be obtained through the use of additional stains to identify other cell populations. Intravenous injection of PE-conjugated anti-F4/80 rapidly labels Kupffer cells, the liver resident macrophage population (Fig. 4A, B), and allows for visualization of platelet interactions with these cells. In livers of LPS treated CD41-YFPki/+ mice co-stained for neutrophils and Kupffer cells, platelets can be seen to adhere to neutrophils, Kupffer cells, regions of the hepatic sinusoids devoid of neutrophils and Kupffer cells, and to areas of co-localization of neutrophils and Kupffer cells, suggesting no single cell type is responsible for all platelet adhesion in the liver (Fig. 4Aiv, Biv, Movie S11). Enumeration of bound platelets demonstrates a significant increase in adherence to neutrophils, Kupffer cells, and to liver sinusoidal endothelium following LPS exposure (Fig. 4C). Inter- estingly, a far greater number of platelets are found to be associated with Kupffer cells than are seen associated with neutrophils, suggesting that Kupffer cells play a central role in platelet aggregation within the liver, an observation to our knowledge never previously noted. Although this finding is novel, it is not entirely surprising given that Kupffer cells occupy a greater area than do neutrophils in any given field of view (Fig. S8), and as such have the capacity to recruit a greater number of platelets. Taken together, the data generated in the CD41-YFPki/+ mice support the observations made using anti-CD49b to visualize platelets and validate this molecule for in vivo labelling of the total peripheral platelet population in mice. Both approaches for visualizing platelets resulted in an approximately 4-fold increase in platelet adhesion ($10 mm2, Fig. 2Di, 3C) with many of the recruited platelets appearing to be associated with adherent neutrophils within the liver. Furthermore, as platelet recruitment to the liver was comparable between the two models, one can PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e25109