Figure 1
Figure 1. Analysis of putative mitochondrial polarization in target cells during cytotoxic lymphocyte induced death. (A) HeLa cells expressing cytochrome c–green fluorescent protein (GFP) and stained with MitoTracker Red (150 nM) were mixed with KHYG1, human NK cells and images were taken every 2 minutes using a 63×/1.30 NA glycerol objective lens on a Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany). Representative images showing (i) frame 1, (ii) KHYG1 interacting with the target, (iii) precytochrome c release, (iv) prerounding, and (v) rounding are shown. A video of this cell is available online (see the Supplemental Materials link at the top of the online article). Images were acquired by an LAS AF version 1.7.0 and processed by a MetaMorph version 7.5.5.0 (MDS Analytical Technologies, Torrance, CA). (B) To represent mitochondrial localization in the cell over time the cell was segmented into 4 quadrants (shown in panel A) where quadrant 1 was designated as the quadrant where the effector first made contact. The fluorescence of MitoTracker Red in the target cell was calculated for each frame. To account for photobleaching and random cell movement, the percentage fluorescence in each of the 4 quadrants was calculated as follows: (fluorescence intensity of the quadrant/fluorescence intensity of the entire cell) × 100. The relative fluorescence intensity was then calculated by dividing the percentage fluorescence in each quadrant by the percentage fluorescence for that quadrant in the first frame. (C) To simplify this analysis for multiple cells, we calculated the percentage fluorescence in each quadrant before any cytotoxic lymphocytes cells were added (T = 0) to establish a reference point, the time directly before CL engagement (Pre Hit), the time directly after lymphocyte engagement (Time of Hit), the time directly before the target cell rounded up (Pre rounding), and the time directly after rounding (Round). The fluorescence of each quadrant was calculated relative to that at T = 0. Data for KHYG1-induced death of 2 HeLa cells where the mitochondria move into quadrant 1 and quadrant 4, respectively, is presented. (D) To determine whether the data using KHYG1 cells were generally applicable, we followed HeLa and HeLa-Bcl-2 cells killed by NK cells isolated from human patients, and MC57-Bcl-2 or MS9II-Bcl-2 cells killed by NK cells isolated from C57BL/6 mice. The difference from T = 0 plus or minus SEM was plotted for the number of cells for each effector/target combination (n = number of individual experiements). Similar data were obtained when murine cytotoxic T cells were used as effectors (not shown).

Analysis of putative mitochondrial polarization in target cells during cytotoxic lymphocyte induced death. (A) HeLa cells expressing cytochrome c–green fluorescent protein (GFP) and stained with MitoTracker Red (150 nM) were mixed with KHYG1, human NK cells and images were taken every 2 minutes using a 63×/1.30 NA glycerol objective lens on a Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany). Representative images showing (i) frame 1, (ii) KHYG1 interacting with the target, (iii) precytochrome c release, (iv) prerounding, and (v) rounding are shown. A video of this cell is available online (see the Supplemental Materials link at the top of the online article). Images were acquired by an LAS AF version 1.7.0 and processed by a MetaMorph version 7.5.5.0 (MDS Analytical Technologies, Torrance, CA). (B) To represent mitochondrial localization in the cell over time the cell was segmented into 4 quadrants (shown in panel A) where quadrant 1 was designated as the quadrant where the effector first made contact. The fluorescence of MitoTracker Red in the target cell was calculated for each frame. To account for photobleaching and random cell movement, the percentage fluorescence in each of the 4 quadrants was calculated as follows: (fluorescence intensity of the quadrant/fluorescence intensity of the entire cell) × 100. The relative fluorescence intensity was then calculated by dividing the percentage fluorescence in each quadrant by the percentage fluorescence for that quadrant in the first frame. (C) To simplify this analysis for multiple cells, we calculated the percentage fluorescence in each quadrant before any cytotoxic lymphocytes cells were added (T = 0) to establish a reference point, the time directly before CL engagement (Pre Hit), the time directly after lymphocyte engagement (Time of Hit), the time directly before the target cell rounded up (Pre rounding), and the time directly after rounding (Round). The fluorescence of each quadrant was calculated relative to that at T = 0. Data for KHYG1-induced death of 2 HeLa cells where the mitochondria move into quadrant 1 and quadrant 4, respectively, is presented. (D) To determine whether the data using KHYG1 cells were generally applicable, we followed HeLa and HeLa-Bcl-2 cells killed by NK cells isolated from human patients, and MC57-Bcl-2 or MS9II-Bcl-2 cells killed by NK cells isolated from C57BL/6 mice. The difference from T = 0 plus or minus SEM was plotted for the number of cells for each effector/target combination (n = number of individual experiements). Similar data were obtained when murine cytotoxic T cells were used as effectors (not shown).

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