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).