Measurement of IDO Activity in MSC Cultures

The activity of IDO was determined by the measurement of L-kynurenine in the supernatant of four MSC cultures as described previously (22 (link)). Briefly, MSC were seeded at a density of 100,000 cells/well in a 6 wells plate and cultured for 4 days with or without 50 ng/mL IFN-γ. 30% trichloroacetic acid was added to the supernatant in a 1:3 ratio. Samples were incubated for 30 min at 50°C and spun down at 12,000 rpm for 5 min. Samples were plated in a 96 wells flat bottom plate and diluted 1:1 in Ehrlich reagent [200 mg 4-dimethylaminobenzaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in 10 ml of glacial acetic acid]. Absorbance was read at 490 nm using a Wallac Victor2 1420 multilabel plate reader (Perkin Elmer, Waltham, MA, USA).