(a) Negative selection on RIPmOVA slices (open squares), displayed as the ratio of live OT-I thymocytes relative to live reference thymocytes present within the slice, normalized to no antigen controls (black squares). Data are pooled from 4 (0, 3, 6, 9, and 12 hr timepoints), 2 (24 hour timepoint), or 1 (48 hour timepoint) experiments, with mean and SEM of n = 3 (48hr WT and RIPmOVA), 6 (24hr WT and RIPmOVA), 9 (0hr WT and RIPmOVA), 7 (6hr RIPmOVA), 11 (12hr WT), 12 (3hr and 9 hr WT and RIPmOVA, 6 hr and 12 hr WT) total slices per condition, respectively, where each dot represents an individual slice. ns not significant (p>0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired two-tailed Student’s t-test of WT vs RIPmOVA for each timepoint, or two-way ANOVA with 95% confidence interval with Tukey’s multiple comparisons test to compare RIPmOVA samples across timepoints (horizontal line). (b) Still images from a time-lapse series showing an example of OT-I thymocyte death. OT-I thymocytes were depleted of mature CD8 SP and labeled with Hoechst and SNARF before overlaying on LysMGFP RIPmOVA thymic slices. Slices were imaged by two-photon scanning laser microscopy. A 30 min movie was recorded in the medulla, with the time elapsed since thymocyte entry into the slice shown in white. Arrows indicate the position of the dying thymocyte.