MDSCs undergo apoptosis and express activated caspase3 when treated in vitro with the Fas agonist antibody Jo2. Leukocytes from the blood of tumor-free or tumor-bearing mice were cultured for 24 hours with Fas agonist Jo2 antibody or irrelevant control antibody, harvested, and labeled for Gr1, CD11b, and cleaved caspase3, and the Gr1⁺CD11b⁺ cells were gated and analyzed for cleaved caspase3. (A) Flow cytometric staining of labeled cells from representative individual mice (left) and percentage of CD11b⁺Gr1⁺ cells expressing cleaved caspase3 from 6 mice/group (right). Average +SD of 2 experiments; P values were calculated with the Mann-Whitney test. (B) Jo2 or irrelevant control mAb-treated MDSCs were lysed, and the lysates were electrophoresed on 12% SDS-PAGE gels, transferred to nitrocellulose, and probed for activated caspase3. The blots were stripped and then reprobed for β-actin. (C) Leukocytes from 4T1 tumor-bearing BALB/c mice were treated with Jo2 or irrelevant mAb; stained with DAPI and with mAbs to Gr1 (Alexa700), cleaved caspase3 (FITC), and CD11b (Alexa-647); and analyzed by confocal microscopy. Bar = 10 μm.