Measuring Adenosine and IDO1 Levels in Mice

Levels of adenosine in the mouse sera were measured using an Adenosine Assay Kit (Cell Biolabs, USA). A reaction mixture was prepared by mixing fluorometric probe (1:100), avidin-horseradish peroxidase (1:500), adenosine deaminase (1:500), purine nucleoside phosphorylase (1:10), and xanthine oxidase 1:50 in assay buffer. A control mixture containing the same contents as for the reaction mixture except for adenosine deaminase was prepared also. Adenosine standard solutions and mouse samples (50 μl) were added individually into the wells of a black microtiter plate; then either 50 μl of the reaction or a control mixture was added in duplicate. The plate was incubated (37°C, 15 min) in darkness. The excitation (530–570 nm) and emission (590–600 nm) of each well’s content was determined using a fluorescence microplate reader (BioTekTM SynergyTM H1 Hybrid Multi-Mode Monochromater Fluorescence Microplate Reader). The net relative fluorescence unit (RFU) of each sample (difference between the RFU of the wells added with reaction-mixture and the well added with control mixture of the same sample) was used to extrapolate the adenosine level in the mouse serum from the standard adenosine RFU curve.
The levels of mouse lung IDO1 were measured as described previously (19 (link)) using IDO1-ELISA kit (LifeSpan Biosciences, USA).