(C.I.) was calculated. C.I. is calculated by dividing the distance the cell moved towards the chemoattractant - straight-line distance by the total -curve-line distance the cell moved [41]. Morphological changes and polarization in interstitially migrating leukocytes Morphological changes in interstitially migrating leukocytes were evaluated using RLOT microscopy (objective magnification 406). Randomly chosen single migrating leukocytes were recorded for 5 min after 60 min of chemotactic stimulation performed either by microinjection or intrascrotal injection. Polarization of interstitially migrating leukocytes was analyzed off-line in digitized in vivo microscopy images. The major axis and the minor axis of single interstitially migrating leukocytes were measured. Polariza- tion was determined by measuring the eccentricity of the cell which is equal to the ratio of the major axis of the cell (longest straight line that can be drawn across the cell) and minor axis (longest straight line that can be drawn across the cell at 90u to the major axis) [41]. Leukocytes with eccentricity of $1.2 were considered as polarized [41]. Imaging of the migratory behavior of leukocyte subsets Visualization and quantitative analysis of interstitial migration of monocytes were made in Cx3CR1gfp/gfp mice 60–90 min after microinjection of MCP-1 (115 nM). Cx3CR1gfp/gfp mice exhibiting green fluorescent protein (GFP)-labeled monocytes were used in References 1. Muller WA (2003) Leukocyte-endothelial-cell interactions in leukocyte transmi- gration and the inflammatory response. Trends Immunol 24: 327–334. 2. Ley K, Laudanna C, Cybulsky MI, Nourshargh S (2007) Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 7: 678–689. 3. Vestweber D (2007) Adhesion and signaling molecules controlling the transmigration of leukocytes through endothelium. Immunol Rev 218: 178–196. 4. Nourshargh S, Marelli-Berg FM (2005) Transmigration through venular walls: a key regulator of leukocyte phenotype and function. Trends Immunol 26: 157–165. 5. Weber C, Fraemohs L, Dejana E (2007) The role of junctional adhesion molecules in vascular inflammation. Nat Rev Immunol 7: 467–477. 6. Heit B, Robbins SM, Downey CM, Guan Z, Colarusso P, et al. (2008) PTEN functions to ‘prioritize’ chemotactic cues and prevent ‘distraction’ in migrating neutrophils. Nat Immunol 9: 743–752. 7. Kay RR, Langridge P, Traynor D, Hoeller O (2008) Changing directions in the study of chemotaxis. Nat Rev Mol Cell Biol 9: 455–463. 8. Sumen C, Mempel TR, Mazo IB, von Andrian UH (2004) Intravital microscopy: visualizing immunity in context. Immunity 21: 315–329. 9. Lammermann T, Bader BL, Monkley SJ, Worbs T, Wedlich-Soldner R, et al. (2008) Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453: 51–55. 10. Mempel TR, Moser C, Hutter J, Kuebler WM, Krombach F (2003) Visualization of leukocyte transendothelial and interstitial migration using reflected light oblique transillumination in intravital video microscopy. J Vasc Res 40: 435–441. 11. Wegmann F, Petri B, Khandoga AG, Moser C, Khandoga A, et al. (2006) ESAM supports neutrophil extravasation, activation of Rho, and VEGF-induced vascular permeability. J Exp Med 203: 1671–1677. 12. Tharp WG, Yadav R, Irimia D, Upadhyaya A, Samadani A, et al. (2006) Neutrophil chemorepulsion in defined interleukin-8 gradients in vitro and in vivo. J Leukoc Biol 79: 539–554. 13. Colditz IG, Schneider MA, Pruenster M, Rot A (2007) Chemokines at large: in- vivo mechanisms of their transport, presentation and clearance. Thromb Haemost 97: 688–693. 14. Friedl P, Weigelin B (2008) Interstitial leukocyte migration and immune function. Nat Immunol 9: 960–969. 15. Hickey MJ, Forster M, Mitchell D, Kaur J, De Caigny C, et al. (2000) L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo. J Immunol 165: 7164–7170. 16. Russo TA, Davidson BA, Topolnycky DM, Olson R, Morrill SA, et al. (2003) Human neutrophil chemotaxis is modulated by capsule and O antigen from an extraintestinal pathogenic Escherichia coli strain. Infect Immun 71: 6435–6445. 17. Young RE, Thompson RD, Larbi KY, La M, Roberts CE, et al. (2004) Neutrophil elastase (NE)-deficient mice demonstrate a nonredundant role for NE in neutrophil migration, generation of proinflammatory mediators, and Leukocyte Migration In Vivo this part of the study. The microinjection was performed at a distance of 25–50 mm from the vessel. Interstitial migration of Cx3CR1gfp/gfp-positive cells and Cx3CR1gfp/gfp-negative cells was visualized using the combination of in vivo RLOT and fluorescence microscopy (objective magnification 206) and analyzed in perivenular ROI (100675 mm) on the vessel side ipsilateral to microinjection. Digital in vivo microscopy video sequences were analyzed using Simple PCI software. Single cell tracking of extravasated Cx3CR1gfp/gfp-negative or Cx3CR1gfp/gfp-positive cells (n = 5 in each group) was performed at 60, 70, and 80 min after microinjection of MCP-1 for 5 min, respectively. Statistical analysis Groups were compared with either ANOVA on ranks followed by Student-Newman-Keuls test (multigroup comparison) or t-test (two-group comparison) using SigmaStat statistic program (Jandel scientific, Erkrath, Germany). Mean values6standard error of the mean (SEM) are given. Differences between experimental groups reaching p value,0.05 were considered as significant. Author Contributions Conceived and designed the experiments: AGK AK CAR PB MR FK. Performed the experiments: AGK. Analyzed the data: AGK AK. Contributed reagents/materials/analysis tools: MR. Wrote the paper: AGK AK FK. phagocytosis in response to zymosan particles in vivo. J Immunol 172: 4493–4502. 18. Ley K, Allietta M, Bullard DC, Morgan S (1998) Importance of E-selectin for firm leukocyte adhesion in vivo. Circ Res 83: 287–294. 19. Reichel CA, Rehberg M, Bihari P, Moser CM, Linder S, et al. (2008) Gelatinases mediate neutrophil recruitment in vivo: evidence for stimulus specificity and a critical role in collagen IV remodeling. J Leukoc Biol 83(4): 864–74. 20. Pruenster M, Rot A (2006) Throwing light on DARC. Biochem Soc Trans 34: 1005–1008. 21. Normark S, Normark BH, Hornef M (2001) How neutrophils recognize bacteria and move toward infection. Nat Med 7: 1182–1184. 22. Linhardt RJ, Toida T (2004) Role of glycosaminoglycans in cellular communication. Acc Chem Res 37: 431–438. 23. Middleton J, Patterson AM, Gardner L, Schmutz C, Ashton BA (2002) Leukocyte extravasation: chemokine transport and presentation by the endothelium. Blood 100: 3853–3860. 24. Bardi G, Niggli V, Loetscher P (2003) Rho kinase is required for CCR7- mediated polarization and chemotaxis of T lymphocytes. FEBS Lett 542: 79–83. 25. Carstanjen D, Yamauchi A, Koornneef A, Zang H, Filippi MD, et al. (2005) Rac2 regulates neutrophil chemotaxis, superoxide production, and myeloid colony formation through multiple distinct effector pathways. J Immunol 174: 4613–4620. 26. Sun CX, Downey GP, Zhu F, Koh AL, Thang H, et al. (2004) Rac1 is the small GTPase responsible for regulating the neutrophil chemotaxis compass. Blood 104: 3758–3765. 27. Alblas J, Ulfman L, Hordijk P, Koenderman L (2001) Activation of Rhoa and ROCK are essential for detachment of migrating leukocytes. Mol Biol Cell 12: 2137–2145. 28. Werr J, Xie X, Hedqvist P, Ruoslahti E, Lindbom L (1998) beta1 integrins are critically involved in neutrophil locomotion in extravascular tissue In vivo. J Exp Med 187: 2091–2096. 29. Worthylake RA, Lemoine S, Watson JM, Burridge K (2001) RhoA is required for monocyte tail retraction during transendothelial migration. J Cell Biol 154: 147–160. 30. Redd MJ, Kelly G, Dunn G, Way M, Martin P (2006) Imaging macrophage chemotaxis in vivo: studies of microtubule function in zebrafish wound inflammation. Cell Motil Cytoskeleton 63: 415–422. 31. Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, et al. (2007) Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science 317: 666–670. 32. Wan MX, Wang Y, Liu Q, Schramm R, Thorlacius H (2003) CC chemokines induce P-selectin-dependent neutrophil rolling and recruitment in vivo: intermediary role of mast cells. Br J Pharmacol 138: 698–706. PLoS ONE | www.plosone.org 11 March 2009 | Volume 4 | Issue 3 | e4693