Kynurenine Production Assay for IDO Activity

IDO activity, represented as concentration of kynurenine produced, was analyzed by a spectrophotometric assay reported earlier31 (link). Cell-free spent culture media were harvested from primary macrophage or macrophage/MSC co-cultures and used immediately for assay. To 120 μl of spent medium, 60 μl of 30% trichloroacetic acid [Sigma Aldrich] was added, mixed thoroughly and centrifuged at 8000 g for 5 min at room temperature. From the clarified supernatant, 85 μl was transferred immediately to 96-well plates and 85 μl of 1% Ehrlich reagent prepared in glacial acetic acid [Sigma Aldrich] was added and incubated for 10 min at room temperature. Absorbance was read at 490 nm on a multimode plate reader [Ensight^TM, Perkin Elmer]. Concentrations were determined against a kynurenine standard [Sigma].