CTLs polarize cytotoxic granules toward the area of contact with recognizing CD8⁺ T cells in the absence of TCR specificity. (A) H-2^d/b (F1) CTLs were labeled with LysoTracker Red for the detection of granules and targeted with 2C CD8⁺ T cells for 1 hour at 37°C. Cells were fixed, stained, and visualized with an LSM 510 Laser Scanning Confocal Microscope (Carl Zeiss) and 100× PLAN Apochromat objectives having a numerical aperture of 1.4. The F1 CTL appears on the left of each panel and stains positive for both H-2D^d (blue, merge) and H-2D^b (green, merge). The 2C cell appears on the right and stains positive only for H-2D^b. Granules appear in red in the merged image. Scale bar = 5 μm. (B) Quantitation of CTL granule polarization. Conjugates of H-2^d/b (F1) or H-2^d CTLs with 2C CD8⁺ T cells were prepared as in panel A and were evaluated for the presence of cytotoxic granule polarization. CTLs were scored as polarized when granules clearly accumulated in the area of contact. “%Polarization” refers to the percentage of conjugates in which the CTL is found polarizing its granules toward the contact area with the recognizing T cell. Values shown are the calculated means ± SD of 3 independent experiments, n > 70 in each.