Cytothripsis is biochemically distinct from apoptosis. (A) Scanning electron micrographs of control or patient T cells after migrating in collagen gel matrices. (B) Mitochondrial membrane potentials of patient or control T cells migrating in collagen gels. A decrease in the ratio of red (JC-1 aggregates) to green (JC-1 monomer) is indicative of loss of potential. (C) Proportions of T cells expressing active caspase-3 after migrating in collagen gels for the indicated times. Eight controls and four patients were tested from four experiments. (D and E) Immunoblotting for highly active cleaved caspase-8 (D) and caspase-9 (E) proteins. Lysates were prepared from control or patient T cells that had migrated in medium (0) or collagen gels for the indicated times. The black line shows that intervening lanes were spliced out. (F) Inability to block cytothripsis with the pan-caspase inhibitor zVAD. Annexin V⁺ or PI⁺ staining of migrating cells (nine controls, four patients, from four experiments) was quantitated by flow cytometry after recovery of unstained cells from gels with collagenase. All panels show a positive control in which apoptosis was induced in healthy donor T cells within the collagen gels using anti-FAS antibodies with Protein A cross-linking. Cells were analyzed after migrating within the collagen gels for the indicated times or as described in the Materials and methods. A, B, D, and E show representatives of three tested patients and controls from three experiments. Bars, 10 µm. C and F show means ± SD. Unpaired Student’s t test was performed for C and F (right), and two-way ANOVA was performed for F (left). Statistical significance indicated by *, P < 0.05; ****, P < 0.0001; ns, nonsignificant.