GC-treated monocytes are protected from apoptosis. Monocytes were treated with 10 nM fluticasone (GC) for 2 days or left untreated (Co) and subsequently challenged with 200 nM staurosporine (STS). (A) After 6 hours of STS treatment we stained for annexin V to detect one of the earliest features of apoptosis, externalization of phosphatidylserine. The proportion of annexin V–positive cells in the population of untreated (□) and STS-treated cells (▪) is shown. The bars represent mean and SEM from 5 independent experiments (**P < .01, Student t test). (B) The proportion of nuclei containing hypodiploid DNA was assessed by the Nicoletti assay after 6 hours of culture without (□) or with (▪) STS. Mean values and SEM from 5 independent experiments are shown (**P < .01, Student t test). (C) To measure changes in intracellular H2O2, control monocytes (□) and GC-treated monocytes (▪), which were treated with STS for the indicated time periods, were labeled for 15 minutes with DHR123. After washing in PBS, fluorescence was measured immediately by flow cytometry. Data are presented as an N-fold increase in fluorescence intensity between unstimulated and STS-simulated cells defined by MFI with STS stimulation/MFI without STS stimulation. Data are shown as the mean and SEM from 3 independent experiments (*P < .05, Student t test). (D) Changes in the level of intracellular glutathione during STS-induced apoptosis were measured at different time points in lysates from untreated (□) and STS-treated (▪) cells fluorimetrically using monochlorobimane. Data are expressed as the percentage of glutathione levels in untreated cells (indicated as 100%). Data are the mean and SEM from 3 independent experiments (*P < .05, Student t test).
