Neutrophil degranulation assay.

Neutrophils were isolated from human peripheral blood samples and degranulation was quantified as described previously (30). Briefly, 10⁶ isolated human neutrophils were infected in triplicate with 10⁷ CFU of wild-type, ΔbipA, or ΔpCD1 Y. pestis CO92 or mock-inoculated with PBS. Neutrophils and Y. pestis were incubated for 1 h at 37°C. After incubation, neutrophils were washed in PBS with 2% FBS, then stained for 25 min at 4°C with CD63-phycoerythrin-Cy7 (clone H5C6, Invitrogen) as a marker of primary granule exocytosis (21). Neutrophils were washed once in 2% FBS in PBS containing propidium idodide (Invitrogen) to exclude dead cells from analysis. Neutrophils were acquired using a Millipore Guava 6HT flow cytometer and analyzed with the InCyte EasyCyte v3.1 software.