Migration chambers were incubated at 37°C for 1 h prior to time-lapse imaging to allow for sedimentation and were then transferred to the microscope
(DM IL, Leica) connected to a digital camera (TP-505D, Topica). Images were taken every 20 s at a magnification of 20× for 3 h using an automated software (Time controlled Recorder Tetra V. 126.96.36.199, SVS-Vistek). To provide adequate culturing conditions (37°C), a thermal measurement feedback regulator (STATOP-4849, Chauvin Arnoux) was connected to an infrared heat lamp (Beurer). Time-lapse movie sequences were analyzed for speed (excluding non-moving periods) and covered distance of migrated cells with a custom build software
(Autocell, IWR-1 datasheet Department of Selleck ABT-263 Dermatology, University of Wuerzburg). The murine experiments were statistically analyzed with an unpaired, two-tailed Student’s t-test. The human experiments were analyzed with a repeated measures, non-parametric Friedman Test and a Dunn’s Multiple Comparison Test as post test. Significance is indicated as *=p<0.05 and **=p<0.01. The authors would like to thank Professor P. Friedl for providing materials, Julia Schlingmann and Heike Menzel for the collection of clinical samples and Michaela Karches-Böhm for excellent technical help. The authors are grateful to all patients and HD for enabling this study. This Sirolimus supplier study is supported by the BMBF Competence Network of MS (UNDERSTANDMS, Alliance “Immunoregulatory networks in MS,” to H. W.). Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They
are made available as submitted by the authors. “
“Abramson Family Cancer Center, Perelman School of Medicine University of Pennsylvania, Philadelphia, PA, USA Two-dimensional (2D) kinetic analysis directly measures molecular interactions at cell–cell junctions, thereby incorporating inherent cellular effects. By comparison, three-dimensional (3D) analysis probes the intrinsic physical chemistry of interacting molecules isolated from the cell. To understand how T-cell tumor reactivity relates to 2D and 3D binding parameters and to directly compare them, we performed kinetic analyses of a panel of human T-cell receptors (TCRs) interacting with a melanoma self-antigen peptide (gp100209–217) bound to peptide-major histocompatibility complex in the absence and presence of co-receptor CD8.