Figure 2.

Ot infection triggered neutrophil activation and apoptosis. Bone marrow-derived neutrophils were prepared by using anti-Ly6G magnetic beads. The purity of BM neutrophils is 96% based on flow cytometric analysis. Cells were infected with Ot (MOI: 10) in vitro and harvested at indicated time-points. (A) The viability of cells was determined by using fixable live/dead viability dye. (B) The percentages of dead neutrophils at various timepoints were presented based on the flow cytometric analysis. (C) Cells were cultured in vitro, infected with Ot and harvested at 4, 10 and 18 hours (h) post-infection. The transcript levels of activation (TNF-α, CXCL10, ICAM-1, iNOS and Arg-1), apoptosis (Caspase-3) and granules (MPO, Proteinase-1 and Elastase) were detected using qRT-PCR. (D) B6 mice (n=4/group) were i.d. infected with CFSE-labeled Ot (4×10³ FFU) in the ears. Mock mice (n=4/group) were i.d. injected with PBS as a control. Skin tissues were harvested at 18 h, digested by collagenase for preparation of a single cell suspension, and then analyzed by flow cytometry. Neutrophils from mock (black) and infected (red) groups were first gated as CD45⁺Ly6G⁺ cells (left panel), and quantified (right panel). Neutrophils were further sorted for Annexin V expression on the cell surface (left panel), and quantified (right panel). Finally, neutrophils were sorted by CFSE expression (Ot-infected) and Annexin V expression (apoptosis) simultaneously (left panel), and quantified (right panel). Values are shown as mean ± SEM from single experiments and are representative of two independent experiments. A two-tailed Student’s t-test was used for the comparison of two groups. *, p<0.05; **, p<0.01; ***, p <0.001; ****, p < 0.0001.