Characterization of ROS responses in GFP-PX-RAW cells in response to S aureus and E coli. RAW264.7 cells expressing a GFP-PX probe for PtdIns(3)P (GFP-PX-RAW cells) were prepared as described in “Preparation of cells.” (A) Cells (5 × 10⁵/well) were added to 1 × 10⁷ unopsonized (▵) or serum-opsonized (■) S aureus (i) or E coli (ii), and chemiluminesence recorded as described in Figure 1. ◇ represent ROS generation in the absence of bacterial addition. Incubations were performed in duplicate, and data (mean ± range) from one representative experiment of 3 are shown, and are expressed as relative light units/sec (RLU/s). (iii) GFP-PX-RAW cells were preincubated with 0-100 nM wortmannin in the presence of luminol as described in “Measurement of ROS production.” Cells (5 × 10⁶/well) were added to 10⁷ serum-opsonized S aureus (■) or E coli (), and light emission measured over 40 minutes as described in Figure 1. All incubations were performed in duplicate and data from at least 3 experiments (mean ± SEM, n ≥ 6), are accumulated light emission, expressed as a percentage of response in the absence of wortmannin. (B) GFP-PX-RAW cells were preincubated for 10 minutes at 37°C with either DMSO vehicle control or 100 nM wortmannin, 0.1 μM TGX221, 3 μM YM-024, 3 μM IC87114 as indicated, alone or in combination, or in the presence of 1 μM AS252424, before addition of serum-opsonized S aureus (■) or E coli (). ROS generation was measured as described in Figure 1. Data are mean plus or minus SEM (n ≥ 6) and are expressed as a percentage of WT untreated responses.