The total activity of IDO1, IDO2, and TDO was evaluated by measuring the levels of Trp and Kyn by HPLC.6 (link)Blood samples were collected in lithium heparin or ethylene diamine tetraacetic acid vacutainer venous blood collection tubes. The serums were separated from blood samples by centrifugation at 3000 g for 15 min and stored at −80 °C.
The serums and cell culture supernatants were treated with 5% perchloric acid and methanol to remove protein, and the supernatants were subjected to HPLC analysis. The analysis was performed on an Agilent 1260 series HPLC system (Agilent Technologies, USA) equipped with a quaternary pump and a UV detector. HPLC analysis of the samples was performed using an Agilent C18 column (5 -μm particle size, L × I.D. 25 cm × 4.6 mm) preceded by a C18 guard column (Dikma, China). The mobile phase (pH 3.6) consisted of 15 mmol/L acetic acid–sodium acetate buffer and acetonitrile at a ratio of 94:6. The detected wavelengths were 280 nm for Trp and 360 nm for Kyn.
The serums and cell culture supernatants were treated with 5% perchloric acid and methanol to remove protein, and the supernatants were subjected to HPLC analysis. The analysis was performed on an Agilent 1260 series HPLC system (Agilent Technologies, USA) equipped with a quaternary pump and a UV detector. HPLC analysis of the samples was performed using an Agilent C18 column (5 -μm particle size, L × I.D. 25 cm × 4.6 mm) preceded by a C18 guard column (Dikma, China). The mobile phase (pH 3.6) consisted of 15 mmol/L acetic acid–sodium acetate buffer and acetonitrile at a ratio of 94:6. The detected wavelengths were 280 nm for Trp and 360 nm for Kyn.
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