Figure 5.

Wortmannin and PD098059 do not inhibit IL-8 stimulated calcium mobilization. Human neutrophils were loaded with 5 μM Indo-1 AM for 1 hr and simultaneously incubated with either carrier alone (A), 100 nM wortmannin for the final 10 min (B) or 50 μM PD098059 for 1 hr (C). [Ca²⁺]_i was determined by flow microfluorimetry using a cytofluorograf 50H (Ortho Diagnostics) with acqcyte software (Phoenix Flow Systems, San Diego). Samples were analyzed for 2 min prior to stimulation to establish the basal [Ca²⁺]_i. IL-8, 25 nM in KRPD-HSA was then added to the sample, indicated by the arrow, and data collection was continued for an additional 13 min. The data was analyzed using mtime software (Phoenix Flow Systems). The data shown are representative of five independent experiments. The increased resting levels of calcium in B and C are due to incomplete removal from the flow cytometer of IL-8 used to stimulate the previous sample as determined by analyzing the samples in different orders (data not shown).