2.5. Detection of Cytokines in Mouse Serum

Blood was collected from the tails of mice each week without anticoagulants, centrifuged for 5 min at 8000 rpm, and the serum was then removed and stored at –30°C. Cytokine levels were measured using the LEGENDplex Mouse Inflammation Panel and the Th Cytokine Panel (BioLegend) according to the manufacturer's instructions. Samples were first diluted 2-fold. Then, 25 μL of assay buffer and 25 μL of the diluted sample or standards with 25 μL of mixed beads were added to each well of a culture plate. The plate was protected from light and agitated (800 rpm for 2 h) at room temperature and then rinsed twice with a washing buffer. Then, 25 μL of the antibody solution was added to each well and the plate was agitated (800 rpm for 1 h). Then, 25 μL of streptavidin phycoerythrin (SA-PE) was added to each well, the plate was agitated (800 rpm for 30 min) and washed, and the beads were resuspended. The samples were then detected using a Beckman Coulter-CytoFLEX flow cytometer. FCS files were analyzed using LEGENDplex Data Analysis software.