Granule-mediated death by cytotoxic lymphocytes does not require mitochondrial polarization toward the immunologic synapse in target cells.

Itohara, Institute of Physical and Chemical Research, Brain Science Institute, Wako, Japan; 100% C57Bl6) and MMP-12 (gift from S. Shapiro, Harvard Medical School and Massachusetts General Hospital, Boston, MA; 50% C57Bl6 50% Swiss) were used. For all experiments, age-, sexand strainmatched littermate mice were used. Mice were maintained in high-efficiency particulate air (HEPA)–filtered individually ventilated cage (IVC) units. All experiments were performed according to the guidelines for care and use of laboratory animals approved by the institutional ethical animal care committee. Mice were injected with an intravenous bolus of 5-FU (200 mg/kg; Fluroblastin; Pfizer SA/NV, Brussels, Belgium). Peripheral blood was repetitively sampled by retroorbital puncture under light anesthesia, and full blood counts (ethylenediaminetetraacetic acid [EDTA]–buffered) were determined on a hemocytometer (Cell-Dyn 1300; Abbott, Abbott Park, IL). Doxycycline was administered via drinking water protected from light (30 mg/kg), as described.2 Mice were killed by cervical dislocation. The femurs were removed, fixed in 2% paraformaldehyde in phosphate-buffered saline for 24 hours, and decalcified in 0.5 M EDTA solution for 8 days. After dehydration and paraffin embedding, 10m longitudinal sections were prepared on Superfrost Plus slides (Thermo Scientific, Braunsweig, Germany). Immunohistochemistry was performed using antibodies against MMP-2 (Santa Cruz Biotechnology, Santa Cruz, CA) and MMP-12 (R&D Systems, Minneapolis, MN). Specificity for MMP staining was performed using deficient mice (not shown). Corresponding secondary antibodies labeled with horseradish peroxidase or biotin for signal amplification via tyramide signal amplification (TSA; PerkinElmer, Waltham, MA) or via Vectastain ABC kit (Vector Laboratories, Burlingame, CA) were used. For light microscopy, sections were developed with 3,3 -diaminobenzidine (DAB; Sigma-Aldrich, Bornem, Belgium) as a chromogen substrate and counterstained with Harris hematoxylin. Analysis was performed on a Zeiss Axioplan2 connected to a 3CCD video camera (DXC-93OP; Sony, Londerzeel, Belgium), and KS300 software (Zeiss, Zaventem, Belgium). We used SPSS version 11.0 for statistical calculations. Unless stated otherwise, data (mean SEM) were statistically analyzed by an unpaired Student t test. To determine the genotypic differences in WBC counts after 5-FU, an ANOVA for repeated measurements was used, complemented with t test to identify statistically significant genotypic differences at each individual time point. A P value less than .05 was considered statistically significant.