Flow cytometry
The spleens were removed under aseptic conditions at the different phase of disease (onset, peak and recovery), and splenocytes were harvested after lysing red blood cells. The spleen mononuclear cells (MNCs) with 1×106 resuspended in 100 ul PBS 1% bovine serum albumin (BSA) were first stained with F4/80, CD11b, CD206, Arg-1, and CD40, then fixed and permeabilized with the Fixation/Permeabilization Solution Kit for 20 minutes (min) (BD PharmingenTM), and then the stained with antibody for intracellular inductible nitric oxide synthase (iNOS).
The cultured macrophages were harvested and resuspended at 1×106 cells in PBS, then stained by F4/80-PE-CY7 (Biolegend), CD11b-FITC (BD PharmingenTM), CD206-BV650 (Biolegend), Arg-1-APC (R&D) for 45 min at room temperature (RT). Flow cytometric data were acquired using a FACS Aria™ flowcytometry (BD Biosciences) and analyzed with FlowJo software version 7.6.1 (flowjo.com).