⁵¹Cr-labeled OTI CTLs demonstrate survival of the killer cell, and target cell death is required for rapid detachment of the killer cell. (A and B) OTI (closed) or OTI.Prf^−/− (open) CTLs were incubated for 5 h with MC57 target cells pulsed with 1 µM OVA₂₅₇ peptide (or with no peptide as a control). Either the CTLs (A) or MC57 cells (B) were labeled with ⁵¹Cr to determine ⁵¹Cr release. Shown is the percentage of specific ⁵¹Cr release as the mean ± SD of two independent pooled experiments. (C) Time from calcium flux to killer cell detachment from target cells. OTI, OTI.GzmAB^−/−, or OTI.Prf^−/− CTLs labeled with fluo-4–AM were added to MC57-OVA₂₅₇ target cells in the absence or presence of caspase inhibitor QVD. After conjugate formation, time was measured from first Ca²⁺ flux in CTL until its detachment from the target. Each data point represents one conjugate, and means are 8.5 ± 0.7, 38.1 ± 5.7, 41.2 ± 7.7, and 41.6 ± 4.4 min (SEM; n = 53, 26, 26, and 66, respectively). (D) Mouse NK cells (WT B6 or GzmAB^−/− or Prf^−/−) were labeled with fluo-4–AM and added to MC57 target cells. Shown is the time from first calcium flux until detachment from the target: 12.77 ± 1.1, 30.2 ± 3.7, and 67.66 ± 5.3 min (SEM; n = 51, 36, and 34, respectively), where each point represents a single NK cell–target cell conjugate. All data points shown in yellow indicate CTL–target cell conjugates that were still attached at the end of the video and therefore underestimate the duration of attachment. Statistical significance was determined by Tukey’s post-hoc ANOVA. *, P < 0.05.