Mouse Gr-1⁺ granulocytes do not express gzmA or gzmB intracellularly. All indicated cell populations were stained, intracellularly, with either rabbit αmgzmAIS followed by FITC-labeled αrabbit IgG (1:500) or with APC-labeled αhugzmB mAb; rabbit IgG and mouse IgG were used as isotype control, as described in “Flow cytometry.” Analysis was performed using FACSCalibur. (A) 1.3E6SN, EL4.F15:αmgzmA IS (1:1000), αhugzmB mAb (1:100). (Bi) Ex vivo-derived LCMV-immune (day 8 after infection) spleen cells from B6, gzmA^-/-, and gzmB^-/- mice were stained with αCD8 mAb (1:100) and, subsequently, with either αmgzmA IS (1:100), or αhugzmB (1:1000) mAb for intracellular expression of gzms and analyzed as described in “Flow cytometry.” (Bii) In vitro-propagated alloreactive (H-2b anti-H-2d; third stimulation) T cells from B6, gzmA^-/-, and gzmB^-/- mice were treated as in panel Bi. (Ci) In vitro-generated granulocytes (BM cells, day 9, G-CSF) from either B6, gzmA^-/-, or gzmB^-/- mice were stained with the αGr-1 mAb and, subsequently, with either αmgzmA IS (1:100) or αhugzmB (1:10) mAb for intracellular expression of gzms, as described in “Flow cytometry.” (Cii) Alternatively, in vitro-enriched B6 granulocytes were sensitized to either LPS (1 μg/mL) or Lip-OspA (10 μg/mL) for 8 hours, prior to phenotypic analysis, using αGr-1 mAb and either αmgzmA IS (1:100) or αhugzmb (1:10) mAb for intracellular expression of gzms as described above. Numbers in graphs indicate the percentages of gated cells.