CTL stopping, lengthy conjugate duration and sustained granule polarization precede death of the recognizing cell. Lysis of recognizing T cells by targeted CTLs as demonstrated by live cell video microscopy. Target H-2^d/b (F1) CTLs were loaded with LysoTracker Red, which accumulates in granules (orange in appearance), and added to calcein-loaded 2C CD8⁺ T cells (green). Cells were then subject to live video microscopy with a Deltavision Restoration microscope (Applied Precision Instruments) using a MicroMax 5 MHz cooled CCD camera (Roper Scientific). Image sequences of the time-lapse recording were processed with SoftWoRx (Applied Precision). (A) Wide field microscopy demonstrating killing of recognizing T cells. F1 CTLs and 2C CD8⁺ cells were stained as described. After 2 hours of incubation, marked depletion of the recognizing 2C CD8⁺ cells was observed. (B) Snapshots depict the sequence of events occurring in the targeted CTL, culminating in lysis of the recognizing T cell. Time elapsed from the beginning of stable cell-cell contact is indicated. Top row: the CTL shifts its granules toward the contact area. Bottom row: sequential lysis of the 2 2C cells may be observed. Scale bar = 10μm. (C) The effect of specific recognition of CTL on conjugate duration and lysis. Calcein-loaded 2C CD8⁺ T cells were placed in culture with target H-2^d/b CTLs or with unrecognizable H-2^s CTLs and were then monitored by video microscopy. The durations of CTL-2C contacts were recorded and plotted. Data shown were acquired from several microscopic fields and are from a single experiment representative of 3 experiments.