The maturation stimulus used influences IDO expression and activity. Human monocyte-derived iDCs were cultured in media alone or exposed to distinct maturation stimuli as indicated. (A) The relative expression levels of IDO were measured using quantitative RT-PCR. IDO expression is represented as a ratio of IDO/GAPDH as compared with iDCs (set to a value of 1.0 to normalize the data). Each bar corresponds to the mean of all donors assayed (n = 1-7). Error bars indicate standard error of the mean (SEM). Non-normalized data are reported in Table 1. (B) IDO protein was detected by Western blot in cell extracts using a polyclonal rabbit anti-human IDO Ab. The cell number from which the protein was derived was normalized prior to loading the gel; and Ponceau Red staining of the membrane confirmed that equivalent protein content was being analyzed (data not shown). (C) Following the different culturing conditions, DCs were washed well and incubated for 4 hours in HBSS containing 100 μM tryptophan. Supernatants were harvested, and the concentration of kynurenine was determined. The mean concentration of kynurenine, as measured by HPLC, is represented (n = 1-6). Error bars indicate SEM. Numeric values and the range observed in different donors are reported in Table 1.