ROS generation in human and mouse neutrophils in response to S aureus and E coli. (A) Human peripheral (i,iii), and mouse bone marrow–derived (ii,iv) neutrophils were prepared, primed with TNFα and GM-CSF, and preincubated with luminol as described in “Preparation of cells”, and “Measurement of ROS production.” Cells (5 × 10⁵/well) were added to 10⁷ unopsonized (▵) or serum-opsonized (■) S aureus (i,ii) or E coli (iii,iv), or incubated in the absence of bacteria (◇) and chemiluminesence recorded using a Berthold Microlumat Plus luminometer. Incubations were performed in duplicate, and data (mean ± range) from one experiment representative of 3 are shown and are expressed as relative light units/sec (RLU/s). (B) Primed bone marrow neutrophils derived from C57BL/6J mice (WT) or mice lacking either the β2-integrin common chain CD18 (CD18^−/−), or the Fc receptor γ chain (FcRγ^−/−) were incubated with S aureus or E coli opsonized with 10% serum generated from C57BL/6J (normal) or RAG2/γ_C^−/− (Ab-deficient) mice. Where indicated, normal serum was depleted of antibody before opsonization, by incubation with protein G sepharose, or heat-inactivated (H-I) at 56°C for 1 hour. Cells (5 × 10⁵/well) were added to 10⁷ serum-opsonized S aureus (■), E coli (), or 10 μM fMLP (), and light emission measured over 40 minutes as described in Figure 1A. ROS production in response to E coli opsonized with antibody-depleted serum was not determined (ND). All experiments were performed in duplicate and data (mean ± SEM) are accumulated light emission for a combination of at least 2 experiments, expressed as a percentage of the response in WT mouse neutrophils to 10% normal serum-opsonized bacteria.