Live cell imaging of CTL with targets. (A–C) Time-lapse microscopy of fluo-4–AM-labeled OTI, OTI.Prf^−/−, and OTI.GzmAB^−/− CTLs killing MC57 target cells in the presence of 100 µM PI. OTI (A), OTI.Prf^−/− (B), or OTI.GzmAB^−/− (C) with MC57-OVA₂₅₇ target cells are shown. Images were acquired every 10 s and show fluo-4 (green)/PI (red)/brightfield overlay. Images depict conjugation of the CTL with the target cell and sequential elevations in CTL intracellular Ca²⁺, followed by cytosolic diffusion of PI into the target cell (A and C). Images are representative of 53, 26, and 26 conjugates, respectively. See also Videos 1–3. Graphs beside each montage display the fold change (F/F₀) of the fluo-4 fluorescence over time in the CTL and fold change in PI fluorescence by the target cells. (D) The time from first calcium flux until PI blush into target cell for targets “hit” by OTI or OTI.GzmAB^−/−. Data represent mean ± SD of 41 and 33 conjugates, respectively. Student’s t test showed no statistical difference. (E) Representative live cell imaging of CTLs with targets with internal fluorescence control. The identical experiment to A was performed, with CTLs also labeled with cell trace far-red. Images were acquired every 22 s, and shown is a montage of fluo-4 (green)/PI (red)/brightfield overlay (left panels) and cell trace far-red (corresponding right panels). Images depict conjugation of the CTLs with the target cell and sequential elevations in CTL intracellular Ca²⁺ (green), followed by cytosolic diffusion of PI into the target cell (red). Time above the montage shows minutes:seconds. The first panel shows regions in which fluorescence intensity was measured. See also Video 4. (F) Graph of montage displaying the fold change (F/F₀) of the fluo-4 (red) and cell trace violet (blue) fluorescence over time in the CTL and fold change in PI fluorescence in the target cell. # denotes PI blush. Bars, 10 µm.