Human CTLs display longer synapse duration in the absence of target cell death. Time-lapse microscopy of fluo-4–AM-labeled human CTLs killing Boleth target cells (prepulsed with GILGFVFTL peptide) in the presence of 100 µM PI ± QVD to block caspases. (A) After conjugate formation, time was measured from the first Ca2+ flux in CTL until it detached from the target cell for two human clones. Each point represents a single CTL–target cell conjugate, and all data points in yellow show conjugates that were still attached at the end of the video, therefore underestimating the duration of attachment. The synapse duration was 14 ± 4.8, 42.9 ± 4.4, 15.4 ± 3.2, and 48.2 ± 6.8 min (SEM; n = 8, 12, 14, and 23, respectively). Statistical significance was determined by Tukey’s ANOVA. *, P < 0.05. Images were acquired every 20 s. (B and C) Montage of human CTLs + Boleth targets (B) and with the addition of QVD (C). Fluo-4 (green)/PI (red)/brightfield overlay is shown. Time corresponds to minutes:seconds. See also Videos 5 and 6. Bars, 5 µm.
