visualization of a liver from a C57BL/6 mouse 4 h after LPS treatment (1 mg/kg i.v.). Two neutrophils coated in adherent platelets can be seen circulating through the liver microvasculature (neutrophils enter FOV at t = 4 s and t = 11 s) (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 autofluores- cence to illustrate vessels. Objective: UPLANSAPO 206/0.70. Scale bar, 20 mm. (MOV) Movie S9 Platelet behaviour within the liver of an untreated CD41-YFPki/+ mouse. Intravital visualization of the liver of an untreated CD41-YFPki/+ mouse. Although some platelets are seen to interact with the liver sinusoids and neutrophils, few adherent platelets are observed. Neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue); YFP+ platelets and liver autofluorescence (green). Objective: UPLAN- SAPO 206/0.70. Scale bar, 20 mm. (MOV) Movie S10 Visualization of platelet adhesion in an LPS treated CD41-YFPki/+ mouse. Intravital visualization of YFP+ platelets interacting with, and adhering to neutrophils within the liver of an LPS (4 h, 1 mg/kg i.v.) treated CD41-YFPki/+ mouse. Neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue); YFP+ platelets and liver autofluorescence (green). Objec- tive: UPLANSAPO 206/0.70. Scale bar, 20 mm. (MOV) Movie S11 Platelet interactions with Kupffer cells within the liver of an LPS treated mouse. Visualization of YFP+ platelets interacting with, and adhering to sinusoids, Kupffer cells and neutrophils within the liver of an LPS (4 h, 1 mg/kg i.v.) treated CD41-YFPki/+ mouse. Kupffer cells labelled with PE- conjugated anti-F4/80 (red); neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue); YFP+ platelets and liver autofluorescence (green). Objective: UPLANSAPO 206/0.70. Scale bar, 20 mm. (MOV) Movie S12 Platelet behaviour in arterioles and postcap- illary venules of the brain in a control mouse. Intravital visualization of an arteriole (bright CD31 staining) and postcap- illary venule (dim CD31 staining) in the brain of an untreated C57BL/6 mouse. No adherent neutrophils and few platelet- endothelium interactions 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: Ob- jective: XLUMPPlanFI 206/0.95W. Scale bar, 20 mm. (MOV) References 1. Semple JW, Freedman J (2010) Platelets and innate immunity. Cell Mol Life Sci 67: 499–511. 2. Andonegui G, Kerfoot SM, McNagny K, Ebbert KV, Patel KD, et al. (2005) Platelets express functional Toll-like receptor-4. Blood 106: 2417–2423. 3. Iannacone M, Sitia G, Isogawa M, Marchese P, Castro MG, et al. (2005) Platelets mediate cytotoxic T lymphocyte-induced liver damage. Nat Med 11: 1167–1169. 4. Qu Z, Chaikof EL (2010) Interface between hemostasis and adaptive immunity. Curr Opin Immunol 22: 634–642. 5. Von Hundelshausen P, Koenen RR, Weber C (2009) Platelet-mediated enhancement of leukocyte adhesion. Microcirculation 16: 84–96. 6. Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, et al. (2007) Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 13: 463–469. Intravital Visualization of Platelets Movie S13 Platelet behaviour in arterioles and postcap- illary venules of the brain in an LPS treated mouse. Intravital visualization of an arteriole (bright CD31 staining) and postcapillary venule (dim CD31 staining) in brain of an LPS (4 h, 1 mg/kg i.v.) treated C57BL/6 mouse. Numerous adherent neutrophils, platelet-neutrophil, and platelet-endothelial interac- tions can be seen within the postcapillary venule. 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 S14 Platelet behaviour in postcapillary venules of the cremaster muscle in a control mouse. Postcapillary venule in a C57BL/6 mouse imaged under control conditions 4 h after local injection of the cremaster with saline. Although no interactions between platelets and rolling, or circulating neutro- phils are seen, some short term (touching-down) interactions of the platelets with 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: UPLANFL N 106/0.30W. Scale bar, 40 mm. (MOV) Movie S15 Platelet-neutrophil interactions in postcap- illary venules of the cremaster muscle after local LPS stimulation. Postcapillary venule in a C57BL/6 mice imaged 4 h after local injection of the cremaster with LPS (0.5 mg/kg) in saline. Platelets are seen to be adhering to, and interacting with endothelium and both adherent and circulating neutrophils (white arrow – appears at t = 18 s). 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: XLUMP- PlanFI 206/0.95W. Scale bar, 20 mm. (MOV) Acknowledgments We thank The Live Cell Imaging Facility at the University of Calgary funded by the Canada Foundation for Innovation and P. Colarusso for training and assistance related to spinning-disk confocal microscopy. We also thank the University of Calgary Flow Cytometry Facility and L. Kennedy for their assistance with the flow cytometric analysis of platelets. Author Contributions Conceived and designed the experiments: CNJ PK. Performed the experiments: CNJ CHYW BP. Analyzed the data: CNJ. Contributed reagents/materials/analysis tools: PK. Wrote the paper: CNJ. 7. Smyth SS, McEver RP, Weyrich AS, Morrell CN, Hoffman MR, et al. (2009) Platelet functions beyond hemostasis. J Thromb Haemost 7: 1759–1766. 8. Brill A, Fuchs TA, Chauhan AK, Yang JJ, De Meyer SF, et al. (2011) von Willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models. Blood 117: 1400–1407. 9. Vowinkel T, Anthoni C, Wood KC, Stokes KY, Russell J, et al. (2007) CD40- CD40 ligand mediates the recruitment of leukocytes and platelets in the inflamed murine colon. Gastroenterology 132: 955–965. 10. Secor D, Li F, Ellis CG, Sharpe MD, Gross PL, et al. (2010) Impaired microvascular perfusion in sepsis requires activated coagulation and P-selectin-mediated platelet adhesion in capillaries. Intensive Care Med 36: 1928–1934. 11. Echtler K, Stark K, Lorenz M, Kerstan S, Walch A, et al. (2010) Platelets contribute to postnatal occlusion of the ductus arteriosus. Nat Med 16: 75–82. PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e25109