Figure 3
Figure 3. Rapid diffusion through repairable pores at the immunological synapse. CL-mediated diffusion of dyes into target cells at the immunological synapse. (A) Time-lapse microscopy of primary human NK cells with HeLa target cells in the presence of 100 μM PI. Images were acquired every 20 seconds and show PI(red)/Brightfield overlay (top) and PI pseudocolor (bottom; color bar indicates the pixel intensity). White arrow indicates the NK effector cell. Images depict cytosolic diffusion of PI into the target cell from the point of NK contact (0 seconds), followed by uniform cytosolic dispersion of PI (40 seconds), cell rounding (7 minutes), membrane blebbing (1 hour, 24 minutes), and secondary necrosis (3 hours, 55 minutes). Scale bar, 10 µm. Cross-sectional line scan analysis: graph shows the cross-sectional diffusion of PI fluorescence (pixel intensity) at different times from the site of NK contact (point X) to the distal point of the target cell (point Y). Cell image from the top panels has been rotated for presentation purposes. Images are representative of 5 to 10 cells from each of 5 independent experiments (using 5 different human donors). See also supplemental Movie 1. (B) Time-lapse microscopy of mouse OT-I cytotoxic T-lymphocytes and MC57-OVA257 targets in the presence of 100 μM PI. Images were acquired every 20 seconds and show PI(red)/Brightfield overlay (top) and PI pseudocolor (bottom; color bar indicates the pixel intensity). White arrow indicates the CTL effector cell. Images depict cytosolic diffusion of PI into the target from the point of CTL contact (0 seconds), followed by cytosolic dispersion of PI (40 seconds), cell rounding (2.5 minutes), membrane blebbing (4 minutes), and late stages of cell death (34 minutes). Scale bar, 10 µm. Images are representative of 5 to 10 cells from each of 5 independent experiments. (C) Time-lapse microscopy of mouse a OT-I cytotoxic T-lymphocyte and a SIINFEKL-pulsed MC57 target in the presence of 100 μM PI and 2.5 μM SYTO 24 green nuclear dye. Images were acquired every 20 seconds and show the SYTO-green(green)/PI(red)/Brightfield overlay. White arrow indicates the CTL effector cell. Images depict cytosolic diffusion of PI into the target from the point of CTL contact (0 seconds), followed by cytosolic dispersion of PI (20 seconds), cell rounding (2.7-3 minutes), membrane blebbing (8 minutes), and late stages of death (23 minutes). Scale bar, 10 µm. Images are representative of 5 to 10 cells from 2 independent experiments. See also supplemental Movie 2. (D-E) Monitoring the PI fluorescence change over time in bystander (non-hit HeLa) and target cells proximal and distal to the point of NK conjugation. Images were acquired every 5.5 seconds. Data represent the mean ± standard error for fold change (F/F0) in PI fluorescence over time (n = 12 target cells and n = 12 non-hit HeLa cells).

Rapid diffusion through repairable pores at the immunological synapse. CL-mediated diffusion of dyes into target cells at the immunological synapse. (A) Time-lapse microscopy of primary human NK cells with HeLa target cells in the presence of 100 μM PI. Images were acquired every 20 seconds and show PI(red)/Brightfield overlay (top) and PI pseudocolor (bottom; color bar indicates the pixel intensity). White arrow indicates the NK effector cell. Images depict cytosolic diffusion of PI into the target cell from the point of NK contact (0 seconds), followed by uniform cytosolic dispersion of PI (40 seconds), cell rounding (7 minutes), membrane blebbing (1 hour, 24 minutes), and secondary necrosis (3 hours, 55 minutes). Scale bar, 10 µm. Cross-sectional line scan analysis: graph shows the cross-sectional diffusion of PI fluorescence (pixel intensity) at different times from the site of NK contact (point X) to the distal point of the target cell (point Y). Cell image from the top panels has been rotated for presentation purposes. Images are representative of 5 to 10 cells from each of 5 independent experiments (using 5 different human donors). See also supplemental Movie 1. (B) Time-lapse microscopy of mouse OT-I cytotoxic T-lymphocytes and MC57-OVA257 targets in the presence of 100 μM PI. Images were acquired every 20 seconds and show PI(red)/Brightfield overlay (top) and PI pseudocolor (bottom; color bar indicates the pixel intensity). White arrow indicates the CTL effector cell. Images depict cytosolic diffusion of PI into the target from the point of CTL contact (0 seconds), followed by cytosolic dispersion of PI (40 seconds), cell rounding (2.5 minutes), membrane blebbing (4 minutes), and late stages of cell death (34 minutes). Scale bar, 10 µm. Images are representative of 5 to 10 cells from each of 5 independent experiments. (C) Time-lapse microscopy of mouse a OT-I cytotoxic T-lymphocyte and a SIINFEKL-pulsed MC57 target in the presence of 100 μM PI and 2.5 μM SYTO 24 green nuclear dye. Images were acquired every 20 seconds and show the SYTO-green(green)/PI(red)/Brightfield overlay. White arrow indicates the CTL effector cell. Images depict cytosolic diffusion of PI into the target from the point of CTL contact (0 seconds), followed by cytosolic dispersion of PI (20 seconds), cell rounding (2.7-3 minutes), membrane blebbing (8 minutes), and late stages of death (23 minutes). Scale bar, 10 µm. Images are representative of 5 to 10 cells from 2 independent experiments. See also supplemental Movie 2. (D-E) Monitoring the PI fluorescence change over time in bystander (non-hit HeLa) and target cells proximal and distal to the point of NK conjugation. Images were acquired every 5.5 seconds. Data represent the mean ± standard error for fold change (F/F0) in PI fluorescence over time (n = 12 target cells and n = 12 non-hit HeLa cells).

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