The synchronization of effector cell activation and target cell rounding relative to target cell PI uptake. Target cells rapidly round following uptake of PI. (A) Frequency distribution of the times (minutes) between the PI blush to the first sign of cell rounding. Columns represent 2-minute time intervals (n = 186 apoptotic target cell deaths from 3 independent human donors). Effector cell activation precedes target cell PI uptake. (B) Time-lapse microscopy of 1 µM Fluo-4-AM–labeled human NK cells with HeLa target cells. Images depict an increase in NK Fluo-4 fluorescence on attachment to the HeLa target (24 seconds), prior to PI uptake by the target (3 minutes, 12 seconds), and subsequent detachment of the effector cell (23 minutes, 12 seconds). Images are representative of 5 to 10 cells from 6 independent experiments (using 6 different human donors). See also supplemental Movie S3. (C) Time-lapse microscopy of 1 µM Fluo-4-AM–labeled wt mouse NK cell with a MC57 target cell showing an increase in NK Fluo-4 fluorescence on attachment to the MC57 target (36 seconds to 1 minute) and prior to PI uptake by the target (1 minute, 36 seconds). Images are representative of 5 to 10 cells from 3 independent experiments. See also supplemental Movie S4. (D) Time-lapse microscopy of 1 µM Fluo-4-AM–labeled pfp^−/− mouse NK cell with a MC57 target cell showing an increase in NK Fluo-4 fluorescence on attachment to the MC57 target (12-24 seconds) but no subsequent PI uptake. Images are representative of 5 to 10 cells from 3 independent experiments. See also supplemental Movie S5. All images (B-D) were acquired every 12 seconds and show Fluo-4(green)/PI(red)/Brightfield overlay (top) and Fluo-4 pseudocolor (bottom; color bar indicates the pixel intensity). White arrows indicate the NK effector cell. Scale bars, 10 µm.