Aminotac 500

Tryptophan, ornithine, and arginine were measured by high-pressure liquid chromatography (HPLC) as indicators of IDO and arginase-1 activity. After a 5-day TIDC and T-cell coculture, the supernatant was collected and tryptophan, ornithine, and arginine concentrations were measured. In brief, 200 μL of cell supernatant was subjected to a deproteinization step using a 30% sulfosalicylic acid solution (Sigma). Then, supernatants were diluted with Jeol sampling buffer (JEOL) containing 0.2 μmol/mL of aminoethyl cysteine and glucosaminic acid (internal standards) (Sigma). Supernatants (50 μL) were then injected into an automated amino acid analyzer (JEOL Aminotac 500) and eluted with lithium citrate buffer. Tryptophan, ornithine, and arginine were detected at 570 nm. Data acquisition and calculations were made using the JEOL Workstation software.