Granzyme B is internalized independently, but requires perforin to induce apoptosis. Jurkat target cells were incubated in medium containing granzyme B for 0 and 60 minutes at 37°C (A). Cells were fixed in 2% paraformaldehyde and immunolabeled with antigranzyme antiserum and staining with Texas Red-conjugated goat antirabbit antibody. Cells were viewed by CLSM. Granzyme B label was not present at 0 minutes, but appeared in a distinct punctate pattern by 60 minutes. Despite the uptake of granzyme B, there were no detectable signs of cell death. (B) Jurkat target cells were treated with granzyme B, washed to remove soluble granzyme, and incubated in sublytic levels of perforin at 37°C for the times indicated. Cells were labeled for DNA fragmentation by the TUNEL protocol with dUTP-FITC. Granzyme B⁺ targets showing the progression of DNA fragmentation observed in apoptotic death induced by granzyme B and perforin from a healthy cell (0 minutes); early DNA fragmentation observed at early time points (30 minutes); an apoptotic cell with considerable DNA fragmentation and reduced nuclear size (60 minutes); and advanced DNA fragmentation and severe nuclear condensation at 120 minutes. (C) Yac-1 targets were treated with granzyme B and buffer or in combination with perforin. Cells were labeled with the DNA-binding dye DAPI and viewed by fluorescence microscopy. Examples of condensed nuclei of apoptotic cells are indicated by arrows. Scale bar is 10 μm.