, 2005). Central memory cells and naive cells have high expression of CCR7 whereas effector memory cells have low expression of CCR7, the chemokine receptor for CCL21. It is likely that central memory cells are the

most responsive to CCL21 among all the subsets of CD8 T cells in our experiments. This is consistent with the increased speed during interstitial motility of central memory CD8 T cells compared to naive counterparts within intact lymph nodes in the absence RAD001 of any antigen (Chtanova et al., 2009). Memory cells have increased surface levels of LFA1 compared to naive cells, which might contribute to higher responsiveness of central memory CD8 T cells to CCL21 co-adsorbed with ICAM1. We also observed that majority of CD45RO cells make contacts with the substratum, that are at least few microns in size, during CCL21-driven chemokinesis whereas majority of the CD45RA cells do not (Fig. 5b). These contacts are dynamic and discontinuous, similar to those observed previously in pre-activated T cells undergoing fast autonomous motility (Jacobelli et al., 2009). These contacts may also contribute to increased motility of CD45RO+ve cells. The novel findings reported in this study were critically buy SAHA HDAC dependent on integrating motility information with additional information from DIC, reflection and two fluorescence channels. In

the case of comparative analysis of CD45RA and CD45RO subsets, these were distinguished based on differential fluorescent dye labels. The fluorescence information allowed us to compare motility characteristics and reflection footprints of attachment simultaneously. This allowed us to delineate the motility and attachment tendencies of the subsets (Fig. 5b). Further delineation based on whether the cells within the subsets had shown contact footprint allowed us to observe that attachment promotes

motility (Fig. S12). In the case of LFA1 at the contact, its surface density could be related to motility characteristics and reflection footprints of attachment (Fig. 5c). We have brought together several existing approaches in building TIAM. The hybrid approach of edge detection followed by Hough transforms is a widely used approach for pattern recognition. Similarly the two-tier approach of linkage of neighboring Chlormezanone objects in consecutive frames followed by temporal linkage of shorter segments is analogous to a recently introduced approach for single-particle tracking (Jaqaman et al., 2008). Put together, these approaches enable robust detection and tracking of cells. Accurate and comprehensive tracking is critical for developing motion models of cell motility and for characterizing heterogeneity in the motility behavior. Studying cellular heterogeneity has yielded better understanding of underlying mechanisms in other contexts (Altschuler and Wu, 2010). Our observation of an inverse relationship between the speed and turn angle of individual cells is a case in point (Fig.