Leukocyte Migration In Vivo Figure 4. Leukocyte interstitial migration. Interstitially migrating leukocytes were visualized using near-infrared RLOT microscopy (objective magnification 206) and tracked during 5 min in digitized video recordings using an imaging software. A - leukocyte interstitial migration after microinjection of MIP-1a, B - after intrascrotal injection of PAF, C – upon intrascrotal injection of MIP-1a, and D - after microinjection of MIP-1a followed by superfusion of the Rho kinase inhibitor Y27632. a - red numbers on the intravital microscopic images shows the position of each analyzed leukocyte at the beginning of cell tracking; b - location of the same leukocytes at the end of the cell tracking, respectively; c - green lines on intravital microscopic images and colored lines on the panels (d) show the migration tracks of single leukocytes; asterisks show the site of microinjection, scale bar 20 mm. doi:10.1371/journal.pone.0004693.g004 60 min after microinjection of MCP-1. Straight-line migration velocity of neutrophils was comparable between the three analyzed time periods (Fig. 8C). Monocytes, however, did not move toward MCP-1, at least at 60–65 min after microinjection, as indicated by lower levels of curve-line and straight-line migration velocity and directionality (Fig. 8B, C, D). In addition, monocytes displayed a less polarized phenotype at 70 min after microinjection of MCP-1 as compared to neutrophils (Fig 8E), nevertheless these values were not significant. Interestingly, straight-line velocity and directionality of monocytes were slightly increased after 70 min and 80 min after microinjection of MCP-1 as compared to those after 60 min (Fig. 8C, D). These results suggest a delayed chemotactic response of monocytes as compared to neutrophils. Although monocytes displayed a target-oriented character of interstitial migration at 70 and 80 min after microinjection, they migrated slower than neutro- phils as presented by lower curve-line, straight-line migration parameters, directionality and polarization (Fig. 8B–E). Figure 5. Leukocyte motility. Parameters of leukocyte motility such as curve-line migration velocity (A), straight-line migration velocity (B), and directionality (C) were determined in digitized in vivo microscopy video sequences 60 min after microinjection of MIP-1a, PAF or E. coli as well as upon intrascrotal application of PAF (PAF i.s.). Interstitially migrating leukocytes (n = 15) were analyzed during 5 min using SimplePCI software. Parameters of leukocyte motility are presented for the vessel side ipsilateral to the microinjection site (black bars), on the contralateral vessel side (white bars), and after intrascrotal application of PAF – (gray bars); mean6SEM; *p,0.05 vs. saline, 1 p,0.05 vs. PAF i.s., & p,0.05 vs. PAF, $ p,0.05 vs. contralateral side; n = 15; E. coli n = 8. doi:10.1371/journal.pone.0004693.g005 PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4693