Figure S3 Intravenous treatment with LPS results in a reduction in circulating platelets. Circulating platelet counts from untreated and mice treated with 1 mg/kg LPS intravenously for 4 h. ** p = 0.01. (TIF) Figure S4 Flow cytometric analysis of platelets from untreated and LPS-treated mice. Absence of neutrophil associated (GR1) (A) or macrophage associated (F4/80) (B) surface markers on platelets from untreated or LPS-treated mice. Staining for CD18 expression on platelets (C, left panel) and granulocytes (D, right panel) obtained from untreated or LPS-treated mice. (TIF) Figure S5 Effect of neutrophil and Kupffer cell deple- tion on platelet aggregation in the mouse liver in response to LPS treatment. Quantification of the number of platelet aggregates equal to, or larger than the indicated sizes in response to LPS treatment of wild-type mice, neutrophil depleted mice, Kupffer cell depleted mice, and mice depleted of both neutrophils and Kupffer cells. *** p,0.001, ** p,0.01, * p,0.05. (TIF) Figure S6 Circulating platelet-neutrophil aggregates. Time lapse images of a circulating platelet-neutrophil aggregate. The circulating neutrophil is denoted by yellow arrows and the associated platelet aggregate is denoted by white arrows (note: the platelet aggregates appear ahead of the neutrophil due to the sequential capture of individual fluorescent channels). Platelets labelled with PE-conjugated anti-CD49b (red); neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue); green is liver autofluorescence to illustrate vessels. Sequential images represent 2.3 s intervals. Scale bar, 20 mm. (TIF) Figure S7 Flow cytometric analysis of blood from CD41- YFPki/+ mice. Representative histograms from four individual CD41-YFPki/+ mice demonstrating variability in the percentage of YFP+ platelets between animals. Histograms pre-gated on size and for positive PE-conjugated anti-CD41 staining. (TIF) Figure S8 Area occupied by neutrophils and Kupffer cells in the livers untreated and LPS-treated mice. The area corresponding to neutrophil staining (GR1+ staining) and Kupffer cell staining (F4/80+ staining) were measured in 12 representative fields of view (FOV) from untreated mice and mice treated with 1 mg/kg LPS intravenously 4 h earlier. *** p,0.001, n.s. not significant. (TIF) Movie S1 In vivo labelling of peripheral platelets by intravenous injection of PE-conjugated anit-CD49b. Intravital visualization of circulating platelets within the liver microvasculature of an untreated C57BL/6 mouse using spinning- disk confocal fluorescence microscopy. Platelets labelled with PE- conjugated anti-CD49b (red); neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue). Objective: UPLANSAPO 106/0.40. Scale bar, 40 mm. (MOV) Movie S2 Platelet interactions in the liver of a control mouse. Platelets labelled with PE-conjugated anti-CD49b can be seen circulating through the liver sinusoids of an untreated C57BL/6 mouse. These platelets are seen to form frequent, but brief interactions with the sinusoid walls and few adherent neutrophils. A small number of platelets appear to adhere to the liver sinusoids even under control conditions. Platelets labelled Intravital Visualization of Platelets with PE-conjugated anti-CD49b (red); neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue). Objective: UPLAN- SAPO 206/0.70. Scale bar, 20 mm. (MOV) Movie S3 Platelet behaviour in the microvasculature of the ear in a control mouse. Although no interactions between platelets and neutrophils are seen in the ear microvasculature of an untreated C57BL/6 mouse, a small number of brief (touching- down) interactions between platelets and the endothelium can be observed. Platelets labelled with PE-conjugated anti-CD49b (red); neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue); endothelium labelled with Alexa Fluor 488-conjugated anti- CD31 (green). Objective: XLUMPPlanFI 206/0.95W. Scale bar, 20 mm. (MOV) Movie S4 Absence of CD49b labelled platelets in a thromboyte depleted mouse. Liver intravital microscopy in a mouse treated for 18 h with anti-thrombocyte serum (100 ml/ mouse i.v.). Although a CD49b labelled NK cell is visible (white arrow), there is an absence of the small adherent and circulating CD49b+ particles confirming the identity of these objects as platelets. NK cell and platelets labelled with PE-conjugated anti- CD49b (red); neutrophils labelled with Alexa Fluor 647-conjugat- ed anti-Gr-1 (blue). Objective: UPLANSAPO 206/0.70. Scale bar, 20 mm. (MOV) Movie S5 Platelet interactions and adhesion within the liver of an LPS treated mouse. Intravital visualization of a liver from a C57BL/6 mouse 4 h after LPS treatment (1 mg/kg i.v.). Platelets can be seen interacting with the sinusoids and neutrophils that have been recruited to the liver, generating dynamic aggregates and sloughing off into circulation. Platelets labelled with PE-conjugated anti-CD49b (red); neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue). Objective: UPLANSAPO 106/0.40. Scale bar, 40 mm. (MOV) Movie S6 4D imaging of platelet neutrophil interactions within the liver of an LPS treated mouse. Sequential intravital z stacks were imaged within the liver of an LPS (4 h, 1 mg/kg i.v.) treated mouse, compiled and rendered as 3D opacity models using the Volocity software package. Platelets can be seen to be interacting with, and adhering to neutrophils within the liver sinusoids. Platelets were labelled with PE-conjugated anti-CD49b (rendered red); neutrophils were labelled with Alexa Fluor 647- conjugated anti-Gr-1 (rendered blue). Objective: UPLANSAPO 206/0.70. Grid 25.7 mm. (MOV) Movie S7 Dynamic platelet interactions with neutro- phils within the liver of an LPS treated mouse. Platelet interactions and adhesion within the liver of an LPS (4 h, 1 mg/kg i.v.) treated C57BL/6 mouse. Platelets can be seen interacting with, adhering to, and ‘‘flowing’’ across the surface of the neutrophils. These platelets are also observed to from unstable and dynamic aggregates on downstream edge of the cell before tearing off into the circulation (white arrow). Platelets labelled with PE-conjugated anti-CD49b (red); neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue). Objective: UPLANSAPO 206/0.70. Scale bar, 20 mm. (MOV) Movie S8 Circulating platelet-neutrophil aggregates within the liver of an LPS treated mouse. Intravital PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e25109